Flea aminopeptidase nucleic acid molecules

ABSTRACT

The present invention relates to flea serine protease and aminopeptidase proteins; to flea serine protease and aminopeptidase nucleic acid molecules, including those that encode such proteins; to antibodies raised against such proteins; and to compounds that inhibit flea serine protease and/or aminopeptidase activities. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitors as well as the use of such therapeutic compositions to protect a host animal from flea infestation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/326,773, entitled "USE OF PROTEASE INHIBITORSAND PROTEASE VACCINES TO PROTECT ANIMALS FROM FLEA INFESTATION", filedOct. 18, 1994, now U.S. Pat. No. 5,766,609, which is acontinuation-in-part of U.S. patent application Ser. No. 07/806,482,entitled "FLEA MIDGUT-GENERATED ANTIFLEA VACCINES", filed Dec. 13, 1991,which issued as U.S. Pat. No. 5,356,622 on Oct. 18, 1994. Bothapplications are each incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel flea protease proteins and theiruse to reduce flea infestation of animals. The present invention alsorelates to the use of anti-flea protease antibodies and other compoundsthat reduce flea protease activity to reduce flea infestation ofanimals.

BACKGROUND OF THE INVENTION

Fleas, which belong to the insect order Siphonaptera, are obligateectoparasites for a wide variety of animals, including birds andmammals. Flea infestation of animals is of health and economic concernbecause fleas are known to cause and/or transmit a variety of diseases.Fleas cause and/or carry infectious agents that cause, for example, fleaallergy dermatitis, anemia, murine typhus, plague and tapeworm. Inaddition, fleas are a problem for animals maintained as pets because theinfestation becomes a source of annoyance for the pet owner who may findhis or her home generally contaminated with fleas which feed on thepets. As such, fleas are a problem not only when they are on an animalbut also when they are in the general environment of the animal.

The medical and veterinary importance of flea infestation has promptedthe development of reagents capable of controlling flea infestation.Commonly encountered methods to control flea infestation are generallyfocussed on use of insecticides in formulations such as sprays,shampoos, dusts, dips, or foams, or in pet collars. While some of theseproducts are efficacious, most, at best, offer protection of a verylimited duration. Furthermore, many of the methods are often notsuccessful in reducing flea populations on the pet for one or more ofthe following reasons: (1) failure of owner compliance (frequentadministration is required); (2) behavioral or physiological intoleranceof the pet to the pesticide product or means of administration; and (3)the emergence of flea populations resistant to the prescribed dose ofpesticide. Additional anti-flea products include nontoxic reagents suchas insect growth regulators (IGRs), including methoprene, which mimicsflea hormones and affect flea larval development.

An alternative method for controlling flea infestation is the use offlea vaccines to be administered to animals prior to or during fleainfestation. However, despite considerable interest in developinganti-flea reagents, no flea vaccine presently exists.

SUMMARY OF THE INVENTION

The present invention relates to flea serine protease and aminopeptidaseproteins; to flea serine protease and aminopeptidase nucleic acidmolecules, including those that encode such proteins; to antibodiesraised against such proteins; and to compounds that inhibit flea serineprotease and/or aminopeptidase activities. The present invention alsoincludes methods to obtain such proteins, nucleic acid molecules,antibodies, and inhibitors. Also included in the present invention aretherapeutic compositions comprising such proteins, nucleic acidmolecules, antibodies, and/or inhibitors as well as the use of suchtherapeutic compositions to protect a host animal from flea infestation.

One embodiment of the present invention is an isolated flea serineprotease nucleic acid molecule that hybridizes under stringenthybridization conditions with a flea serine protease gene. Particularlypreferred flea serine protease nucleic acid molecules include nucleicacid sequences SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, the nucleic acid sequencesdisclosed in Table 2 and/or nucleic acid sequences encoding proteinshaving amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, and/or the amino acid sequences disclosed in Table 2, as well asallelic variants of any of those nucleic acid sequences.

Another embodiment of the present invention is an isolated fleaaminopeptidase nucleic acid molecule that hybridizes under stringenthybridization conditions with a flea aminopeptidase gene. A particularlypreferred flea aminopeptidase nucleic acid molecule includes nucleicacid sequence SEQ ID NO:50 or an allelic variant thereof.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include flea serine protease and/oraminopeptidase nucleic acid molecules of the present invention. Alsoincluded are methods to produce such nucleic acid molecules, recombinantmolecules, recombinant viruses and recombinant cells.

Another embodiment of the present invention includes an isolated fleaserine protease protein, including a protein that includes a flea serineprotease protein. A preferred flea serine protease protein is capable ofeliciting an immune response against a natural flea protease whenadministered to an animal and/or of having serine protease activity.Particularly preferred flea serine protease proteins are those encodedby preferred flea serine protease nucleic acid molecules of the presentinvention.

Yet another embodiment of the present invention includes an isolatedflea aminopeptidase protein, including a protein that includes a fleaaminopeptidase protein. A preferred flea aminopeptidase protein iscapable of eliciting an immune response against a natural flea proteasewhen administered to an animal and/or of having aminopeptidase activity.A particularly preferred flea aminopeptidase protein is a protein thatincludes SEQ ID NO:51 or a protein that is encoded by a nucleic acidmolecule that is an allelic variant of a nucleic acid moleculecomprising SEQ ID NO:50.

The present invention also relates to mimetopes of flea serine proteaseand aminopeptidase proteins as well as to isolated antibodies thatselectively bind to flea serine protease proteins or mimetopes thereofor to flea aminopeptidase proteins or mimetopes thereof. Also includedare methods, including recombinant methods, to produce proteins,mimetopes and antibodies of the present invention.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of reducing flea infestation. Such atherapeutic composition includes one or more of the following protectivecompounds: an isolated flea serine protease protein or a mimetopethereof; an isolated flea serine protease nucleic acid molecule thathybridizes under stringent hybridization conditions with a flea serineprotease gene; an isolated antibody that selectively binds to a fleaserine protease protein; an inhibitor of flea serine protease activityidentified by its ability to inhibit flea serine protease activity; anisolated flea aminopeptidase protein or a mimetope thereof; an isolatedflea aminopeptidase nucleic acid molecule that hybridizes understringent hybridization conditions with a flea aminopeptidase gene; anisolated antibody that selectively binds to a flea aminopeptidaseprotein; and an inhibitor of flea aminopeptidase activity identified byits ability to inhibit flea aminopeptidase activity. A preferredtherapeutic composition of the present invention also includes anexcipient, an adjuvant and/or a carrier. Also included in the presentinvention is a method to reduce flea infestation. The method includesthe step of administering to the animal a therapeutic composition of thepresent invention.

Another embodiment of the present invention is a method to identify acompound capable of inhibiting flea serine protease or fleaaminopeptidase activity. The method includes the steps of: (a)contacting an isolated flea serine protease protein or a fleaaminopeptidase protein with a putative inhibitory compound underconditions in which, in the absence of the compound, the protein has,respectively, serine protease or aminopeptidase activity; and (b)determining if the putative inhibitory compound inhibits the respectiveactivity. Also included in the present invention is a test kit toidentify a compound capable of inhibiting flea serine protease or fleaaminopeptidase activity. Such a kit includes an isolated flea serineprotease protein having serine protease activity or an isolated fleaaminopeptidase protein having aminopeptidase activity and a means fordetermining the extent of inhibition of the respective activity in thepresence of a putative inhibitory compound.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a protease substrate gel analysis of the relativeproteolytic activity in 1, 2, 5 or 10 midguts from either fed or unfedfemale fleas.

FIG. 2 depicts a protease substrate gel analysis of fed and unfed midgutpreparations incubated in the presence or absence of a serine proteaseinhibitor.

FIG. 3 depicts a protease substrate gel analysis of various fractionsobtained in the preparation of a soluble flea midgut preparationincubated in the presence or absence of a serine protease inhibitor.

FIG. 4 depicts a protease substrate gel showing midgut protease activityas a function of time after flea blood feeding.

FIG. 5A depicts a Coomassie stained SDS-PAGE of partially purified(1,3-³ H)-diisopropylfluoro-phosphate-labeled fed flea midgut serineproteases.

FIG. 5B depicts an autoradiogram of the SDS-PAGE gel of FIG. 5A ofpartially purified (1,3-³ H)-diisopropylfluoro-phosphate-labeled fedflea midgut serine proteases.

FIG. 6 depicts the mean viability of adult (both male and female) fleasfed blood containing certain protease inhibitors.

FIG. 7 depicts the mean fecundity of adult female fleas fed bloodcontaining certain protease inhibitors.

FIG. 8 depicts the mean viability of adult (both male and female) fleasfed blood containing certain protease inhibitors.

FIG. 9 depicts the mean fecundity of adult female fleas fed bloodcontaining certain protease inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes the use of compounds that inhibit fleaprotease activity to protect a host animal from flea infestation. Theinventors have discovered that proteases are significant components ofthe flea midgut and are good targets for immunotherapeutic and/orchemotherapeutic intervention to reduce flea burden both on the hostanimal and in the immediate (i.e., surrounding) environment of theanimal. The inventors have shown, for example, that the viability and/orfecundity of fleas consuming a blood meal is reduced when the blood mealcontains compounds that reduce flea protease activity, probably becausethe compounds interfere with flea digestion and other functions.Compounds that reduce the amount and/or activity of flea proteaseswithout substantially harming the host animal are included in thepresent invention. Such compounds include flea protease vaccines,anti-flea protease antibodies, flea protease inhibitors, and/orcompounds that suppress protease synthesis; such compounds are discussedin more detail below.

One embodiment of the present invention is a method to protect a hostanimal from flea infestation by treating the animal with a compositionthat includes a compound that reduces the protease activity of fleasfeeding (includes fleas in the process of feeding as well as fleashaving fed) from the treated animal thereby reducing the flea burden onthe animal and in the environment of the animal. It is to be noted thatthe term "a" or "an" entity refers to one or more of that entity; forexample, a compound refers to one or more compounds. As such, the terms"a" (or "an"), "one or more" and "at least one" can be usedinterchangeably herein. Thus, a composition of the present invention caninclude one or more compounds that target (reduced the activity of) oneor more proteases in the flea.

As used herein, the phrase "to protect an animal from flea infestation"refers to reducing the potential for flea population expansion on andaround the animal (i.e., reducing the flea burden). Preferably, the fleapopulation size is decreased, optimally to an extent that the animal isno longer bothered by fleas. A host animal, as used herein, is an animalfrom which fleas can feed by attaching to and feeding through the skinof the animal. Fleas, and other ectoparasites, can live on a host animalfor an extended period of time or can attach temporarily to an animal inorder to feed. At any given time, a certain percentage of a fleapopulation can be on a host animal whereas the remainder can be in theenvironment surrounding the animal (i.e., in the environment of theanimal). Such an environment can include not only adult fleas, but alsoflea eggs and/or flea larvae. The environment can be of any size suchthat fleas in the environment are able to jump onto and off of a hostanimal. As such, it is desirable not only to reduce the flea burden onan animal per se, but also to reduce the flea burden in the environmentsurrounding the animal.

In accordance with the present invention, a host animal is treated byadministering to the animal a compound of the present invention in sucha manner that the compound itself (e.g., a protease inhibitor, proteasesynthesis suppressor or anti-flea protease antibody) or a productgenerated by the animal in response to administration of the compound(e.g., antibodies produced in response to a flea protease vaccine, orconversion of an inactive inhibitor "prodrug" to an active proteaseinhibitor) ultimately enters the flea midgut. An animal is preferablytreated in such a way that the compound or product thereof enters theblood stream of the animal. Fleas are then exposed to the compound whenthey feed from the animal. For example, flea protease inhibitorsadministered to an animal are administered in such a way that theinhibitors enter the blood stream of the animal, where they can be takenup by feeding fleas. In another embodiment, when a host animal isadministered a flea protease vaccine, the treated animal mounts animmune response resulting in the production of antibodies against theprotease (anti-flea protease antibodies) which circulate in the animal'sblood stream and are taken up by fleas upon feeding. Blood taken up byfleas enters the flea midgut where compounds of the present invention,or products thereof, such as anti-flea protease antibodies, fleaprotease inhibitors, and/or protease synthesis suppressors, interactwith, and reduce proteolytic activity in the flea midgut. The presentinvention also includes the ability to reduce larval flea infestation inthat when fleas feed from a host animal that has been administered atherapeutic composition of the present invention, at least a portion ofcompounds of the present invention, or products thereof, in the bloodtaken up by the flea are excreted by the flea in feces, which issubsequently ingested by flea larvae. It is of note that flea larvaeobtain most, if not all, of their nutrition from flea feces.

In accordance with the present invention, reducing proteolytic activityin flea midguts can lead to a number of outcomes that reduce flea burdenon treated animals and their surrounding environments. Such outcomesinclude, but are not limited to, (a) reducing the viability of fleasthat feed from the treated animal, (b) reducing the fecundity of femalefleas that feed from the treated animal, (c) reducing the reproductivecapacity of male fleas that feed from the treated animal, (d) reducingthe viability of eggs laid by female fleas that feed from the treatedanimal, (e) altering the blood feeding behavior of fleas that feed fromthe treated animal (e.g., fleas take up less volume per feeding or feedless frequently), (f) reducing the viability of flea larvae, for exampledue to the feeding of larvae from feces of fleas that feed from thetreated animal and/or (g) altering the development of flea larvae (e.g.,by decreasing feeding behavior, inhibiting growth, inhibiting (e.g.,slowing or blocking) molting, and/or otherwise inhibiting maturation toadults).

One embodiment of the present invention is a composition that includesone or more compounds that reduce the activity of one or more fleaproteases directly (e.g., an anti-flea protease antibody or a fleaprotease inhibitor) and/or indirectly (e.g., a flea protease vaccine).Suitable flea proteases to target include flea aminopeptidases, fleacarboxypeptidases and/or flea endopeptidases. Preferred flea proteasesto target include, but are not limited to, serine proteases,metalloproteases, aspartic acid proteases and/or cysteine proteases. Itis to be noted that these preferred groups of proteases includeaminopeptidases, carboxypeptidases and/or endopeptidases. Preferred fleaproteases to target include, but are not limited to, proteases thatdegrade hemoglobin, proteases involved in blood coagulation and/or lytic(anti-coagulation) pathways, proteases involved in the maturation ofpeptide hormones, proteases that inhibit complement or other host immuneresponse elements (e.g., antibodies) and/or proteases involved invitellogenesis. A number of proteases are known to those skilled in theart, including, but not limited to, aminopeptidases, such as leucineaminopeptidase and aminopeptidases B and M; astacin-likemetalloproteases; calpains; carboxypeptidases, such as carboxypeptidasesA, P and Y; cathepsins, such as cathepsins B, D, E, G, H, and L,chymotrypsins; cruzipains; meprins; papains; pepsins; renins;thermolysins and trypsins. A particularly preferred protease to targetis a protease having a proteolytic activity that, when targeted with acomposition of the present invention, reduces flea burden withoutsubstantially harming the host animal. Such a protease can be identifiedusing, for example, methods as disclosed herein.

One aspect of the present invention is the discovery that a substantialamount of the proteolytic activity found in flea midguts is serineprotease activity. Both in vitro and in vivo studies using a number ofprotease inhibitors substantiate this discovery, details of which aredisclosed in the Examples. As such a particularly preferred protease totarget is a serine protease. Examples of serine proteases, include, butare not limited to, acrosins, bromelains, cathepsin G, chymotrypsins,collagenases, elastases, factor Xa, ficins, kallikreins, papains,plasmins, Staphylococcal V8 proteases, thrombins and trypsins. In oneembodiment, a preferred flea serine protease to target includes aprotease having trypsin-like or chymotrypsin-like activity. It isappreciated by those skilled in the art that an enzyme having "like"proteolytic activity has similar activity to the referenced protease,although the exact structure of the preferred substrate cleaved maydiffer. "Like" proteases usually have similar tertiary structures astheir referenced counterparts.

Protease inhibitor studies disclosed in the Examples section alsoindicate that additional preferred proteases to target includeaminopeptidases and/or metalloproteases. Examples of such proteasesinclude exo- and endo-metalloproteases, digestive enzymes, and enzymesinvolved in peptide hormone maturation. One example of an aminopeptidasethat is also a metalloprotease is leucine aminopeptidase.

Suitable compounds to include in compositions of the present inventioninclude, but are not limited to, a vaccine comprising a flea protease (aflea protease vaccine), an antibody that selectively binds to a fleaprotease (an anti-flea protease antibody), a flea protease inhibitor (acompound other than a vaccine or an antibody that inhibits a fleaprotease), and a mixture of such compounds. As used herein, a mixturethereof refers to a combination of one or more of the cited entities.Compositions of the present invention can also include compounds tosuppress protease synthesis or maturation, such as, but not limited to,protease modulating peptides.

A preferred embodiment of the present invention is a flea proteasevaccine and its use to reduce the flea population on and around ananimal. A flea protease vaccine can include one or more proteins capableof eliciting an immune response against a flea protease and can alsoinclude other components. Preferred flea protease vaccines include aflea serine protease, a flea metalloprotease, a flea aspartic acidprotease and/or a flea cysteine protease, with flea serine protease,flea metalloprotease and/or flea aminopeptidase vaccines being morepreferred. Examples of flea protease vaccines include soluble fleamidgut preparations of the present invention as well as one or moreisolated proteins of the present invention.

One embodiment of the present invention is a soluble flea midgutpreparation. Such a preparation includes primarily components naturallypresent in the lumen of a flea midgut and, depending on the method ofpreparation, can also include one or more peripheral midgut membraneproteins. Methods to preferentially include, or exclude, membraneproteins from such a preparation are known to those skilled in the art.The present invention includes the discovery that such a preparation hasproteolytic activity, of which a substantial portion is serine proteaseactivity. Preferably at least about 70 percent of the proteolyticactivity in a soluble flea midgut soluble preparation is serine proteaseactivity, as can be indicated by the ability to inhibit at least about70 percent of the proteolytic activity with4-2-aminoethyl-benzenesulfonylfluoride-hydrochloride (AEBSF). Serineprotease activity can also be identified using other known inhibitors orsubstrates. Other preferred inhibitors that can inhibit at least about70 percent of the proteolytic activity of a soluble flea midgutpreparation of the present invention include soybean trypsin inhibitor,1,3-diisopropylfluoro-phosphate or leupeptin.

A soluble flea midgut preparation of the present invention includesproteases that range in molecular weight from about 5 kilodaltons (kD)to about 200 kD, as determined by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), with at least a substantial portionof the serine proteases ranging in molecular weight from about 5 kD toabout 60 kD, as determined by SDS-PAGE. A substantial portion ofprotease activity in a soluble flea midgut preparation of the presentinvention has a pH activity optimum ranging from about pH 5 to about pH10, preferably an activity optimum ranging from about pH 7 to about pH9, and even more preferably an activity optimum of about pH 8. While notbeing bound by theory, such a pH optimum suggests that a largeproportion of proteases in soluble flea midgut preparations of thepresent invention are serine proteases. It is also interesting to notethat the pH of the flea midgut is also about pH 8. The findings thatproteases in soluble flea midgut preparations of the present inventionexhibit a varied pattern of inhibition by protease inhibitors of a giventype (e.g., serine protease inhibitors), as well as variances seen inmolecular weights and pH optima of the proteases, suggest that there area number of protease isoforms in such preparations.

A soluble flea midgut preparation of the present invention is preferablyprepared by a method that includes the steps of (a) disrupting a fleamidgut to produce a mixture including a liquid portion and a solidportion and (b) recovering the liquid portion to obtain a soluble fleamidgut preparation. Such a method is a simplified version of methodsdisclosed in U.S. Ser. No. 07/806,482, ibid. It is to be noted that inaccordance with the present invention, methods disclosed in U.S. Ser.No. 07/806,482 ibid. can also be used to prepare soluble flea midgutpreparations having similar proteolytic activities.

Flea midguts can be obtained (e.g., dissected from) from unfed fleas orfrom fleas that recently consumed a blood meal (i.e., blood-fed fleas).Such midguts are referred to herein as, respectively, unfed flea midgutsand fed flea midguts. Flea midguts can be obtained from either male orfemale fleas. As demonstrated in the Examples section, female fleamidguts exhibit somewhat more proteolytic activity than do male fleamidguts. Furthermore, fed flea midguts have significantly moreproteolytic activity than do unfed flea midguts. While not being boundby theory, it is believed that blood feeding induces in flea midguts thesynthesis and/or activation of proteases as well as other factors (e.g.,enzymes, other proteins, co-factors, etc.) important in digesting theblood meal, as well as in neutralizing host molecules potentiallydamaging to the flea (e.g., complement, immunoglobulins, bloodcoagulation factors). It is also to be appreciated that unfed fleamidguts may contain significant targets not found in fed flea midgutsand vice versa. Furthermore, although the present application focussesprimarily on flea midgut proteases, it is to be noted that the presentinvention also includes other components of soluble flea midgutpreparations of the present invention that provide suitable targets toreduce flea burden on an animal and in the environment of that animal;see also U.S. Ser. No. 07/806,482, now U.S. Pat. No. 5,328,622 ibid.

Methods to disrupt flea midguts in order to obtain a soluble flea midgutpreparation are known to those skilled in the art and can be selectedaccording to, for example, the volume being processed and the buffersbeing used. Such methods include any technique that promotes cell lysis,such as, but are not limited to, chemical disruption techniques (e.g.,exposure of midguts to a detergent) as well as mechanical disruptiontechniques (e.g., homogenization, sonication, use of a tissue blender orglass beads, and freeze/thaw techniques).

Methods to recover a soluble flea midgut preparation are also known tothose skilled in the art and can include any method by which the liquidportion of disrupted flea midguts is separated from the solid portion(e.g., filtration or centrifugation). In a preferred embodiment,disrupted flea midguts are subjected to centrifugation, preferably at anacceleration ranging from about 10,000×g to about 15,000×g for severalminutes (e.g., from about 1 minute to about 15 minutes). The supernatantfrom such a centrifugation comprises a soluble flea midgut preparationof the present invention.

The present invention also includes an isolated protein that includes anamino acid sequence encoded by a nucleic acid molecule capable ofhybridizing under stringent conditions (i.e., that hybridize understringent hybridization conditions) with a nucleic acid molecule thatencodes a protease present in (i.e., can be found in) a flea midgut,such as a midgut from a blood-fed female flea, a midgut from a blood-fedmale flea, a midgut from an unfed female flea or a midgut from an unfedmale flea. A preferred midgut protease is present in the lumen of themidgut.

An isolated protein of the present invention, also referred to herein asan isolated protease protein, preferably is capable of eliciting animmune response against a flea midgut protease and/or has proteolyticactivity. According to the present invention, an isolated, orbiologically pure, protein, is a protein that has been removed from itsnatural milieu. As such, "isolated" and "biologically pure" do notnecessarily reflect the extent to which the protein has been purified.An isolated protease protein can be obtained from its natural source.Such an isolated protein can also be produced using recombinant DNAtechnology or chemical synthesis.

As used herein, an isolated protein of the present invention can be afull-length protein or any homologue of such a protein, such as aprotein in which amino acids have been deleted (e.g., a truncatedversion of the protein, such as a peptide), inserted, inverted,substituted and/or derivatized (e.g., by glycosylation, phosphorylation,acetylation, myristylation, prenylation, palmitoylation, amidationand/or addition of glycerophosphatidyl inositol) such that the homologuecomprises a protein having an amino acid sequence that is sufficientlysimilar to a natural flea midgut protease that a nucleic acid sequenceencoding the homologue is capable of hybridizing under stringentconditions to (i.e., with) a nucleic acid sequence encoding thecorresponding natural flea midgut protease amino acid sequence. As usedherein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid molecules, includingoligonucleotides, are used to identify similar nucleic acid molecules.Such standard conditions are disclosed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press,1989.

The minimal size of a protein homologue of the present invention is asize sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid with the complementary sequence of a nucleicacid molecule encoding the corresponding natural protein. As such, thesize of the nucleic acid molecule encoding such a protein homologue isdependent on nucleic acid composition and percent homology between thenucleic acid molecule and complementary sequence as well as uponhybridization conditions per se (e.g., temperature, salt concentration,and formamide concentration). The minimal size of such nucleic acidmolecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode a protease proteinhomologue of the present invention is from about 12 to about 18nucleotides in length. There is no limit, other than a practical limit,on the maximal size of such a nucleic acid molecule in that the nucleicacid molecule can include a portion of a gene, an entire gene, ormultiple genes, or portions thereof. Similarly, the minimal size of aprotease protein homologue of the present invention is from about 4 toabout 6 amino acids in length, with preferred sizes depending on whethera full-length, multivalent (i.e., fusion protein having more than onedomain each of which has a function), or functional portions of suchproteins are desired. Protease protein homologues of the presentinvention preferably have protease activity and/or are capable ofeliciting an immune response against a flea midgut protease.

A protease protein homologue of the present invention can be the resultof allelic variation of a natural gene encoding a flea protease. Anatural gene refers to the form of the gene found most often in nature.Protease protein homologues can be produced using techniques known inthe art including, but not limited to, direct modifications to a geneencoding a protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis. Isolated proteaseproteins of the present invention, including homologues, can beidentified in a straight-forward manner by the proteins' ability toeffect proteolytic activity and/or to elicit an immune response againsta flea midgut protease. Such techniques are known to those skilled inthe art.

A preferred protease protein of the present invention is a flea serineprotease, a flea metalloprotease, a flea aspartic acid protease, a fleacysteine protease, or a homologue of any of these proteases. A morepreferred protease protein is a flea serine protease, a fleametalloprotease or a homologue of either. Also preferred is a fleaaminopeptidase or a homologue thereof. Particularly preferred is a fleaserine protease or a homologue thereof.

Preferred protease proteins of the present invention are flea proteaseproteins having molecular weights ranging from about 5 kD to about 200kD, as determined by SDS-PAGE, and homologues of such proteins. Morepreferred are flea protease proteins having molecular weights rangingfrom about 5 kD to about 60 kD, as determined by SDS-PAGE, andhomologues of such proteins. Even more preferred are flea serineprotease proteins and particularly those having molecular weights ofabout 26 kD (denoted PfSP26), about 24 kD (denoted PfSP24), about 19 kD(denoted PfSP19) and about 6 kD (denoted PfSP6), as determined bySDS-PAGE, and homologues of such proteins.

One preferred embodiment of the present invention is an isolated fleaprotease protein that includes an amino acid sequence encoded by anucleic acid molecule that hybridizes under stringent hybridizationconditions with a flea serine protease gene or with a fleaaminopeptidase gene. As used herein, a flea protease gene includes allnucleic acid sequences related to a natural flea protease gene such asregulatory regions that control production of a flea protease proteinencoded by that gene (such as, but not limited to, transcription,translation or post-translation control regions) as well as the codingregion itself.

The inventors have discovered an extensive family of serine proteases,encoded by a family of serine protease genes. Such a gene family may bedue to allelic variants (i.e., genes having similar, but different,sequences at a given locus in a population of fleas) and/or to, theexistence of serine protease genes at more than one locus in the fleagenome. As such, the present invention includes flea serine proteasegenes comprising not only the nucleic acid sequences disclosed herein(e.g., genes including nucleic acid sequences SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38 and/or the nucleic acid sequences disclosed in Table 2) and/ornucleic acid sequences encoding proteins having amino acid sequences asdisclosed herein (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,and/or the amino acid sequences disclosed in Table 2), but also allelicvariants of any of those nucleic acid sequences. (It should be notedthat since nucleic acid sequencing technology is not entirelyerror-free, all sequences represented herein are at best apparent (i.e.,deduced) nucleic acid or amino acid sequences.)

A preferred flea aminopeptidase gene includes nucleic acid sequence SEQID NO:50, which encodes an aminopeptidase protein including SEQ IDNO:51. Additional preferred aminopeptidase genes include allelicvariants of SEQ ID NO:50.

A preferred flea serine protease protein of the present invention isencoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions with at least one of the following nucleic acidmolecules: nfSP1, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8,nfSP9, nfSP10, nfSP11, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16 andnfSP17. As used herein, each of these nucleic acid molecules representthe entire coding region of a flea serine protease gene of the presentinvention. Nucleic acid molecules that contain partial coding regions orother parts of the corresponding gene are denoted by names that includethe size of those nucleic acid molecules (e.g., nfSP4₁₅₆). Nucleic acidmolecules containing apparent full length coding regions for which thesize is known also are denoted by names that include the size of thosenucleic acid molecules (e.g., nfSP4₆₂₇). The production, and at leastpartial nucleic acid sequence, of such nucleic acid molecules isdisclosed in the Examples.

Particularly preferred serine protease proteins are encoded by a nucleicacid molecule that hybridizes under stringent hybridization conditionswith at least one of the following nucleic acid molecules: nfSP4₆₂₇,nfSP1₁₅₆, nfSP2₁₆₈, nfSP3₁₇₇, nfSP4₁₅₆, nfSP5₁₅₉, nfSP6₁₆₈, nfSP7₁₅₉,nfSP8₁₈₆, nfSP9₁₆₈, nfSP10₁₂₀, and nfSP11₁₆₂. Even more preferred serineprotease proteins include the following amino acid sequences: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, SEQID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, and/or the amino acid sequencespresented in Table 2. Additional particularly preferred serine proteaseproteins are encoded by allelic variants of nucleic acid moleculesencoding proteins that include the cited amino acid sequences. Alsopreferred are flea serine protease proteins including regions that haveat least about 50%, preferably at least about 75%, and more preferablyat least about 90% identity with flea serine protease proteins havingamino acid sequences as cited herein.

A preferred flea aminopeptidase protein of the present invention isencoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions with nucleic acid molecule nfAP₄₅₃,theproduction of which is described in the Examples. Even more preferred isan aminopeptidase that includes amino acid sequence SEQ ID NO:51 or anaminopeptidase encoded by an allelic variant of a nucleic acid moleculethat includes SEQ ID NO:50. Also preferred are flea aminopeptidaseproteins including regions that have at least about 50%, preferably atleast about 75%, and more preferably at least about 90% identity withSEQ ID NO:51.

One embodiment of the present invention is an isolated protein havingproteolytic activity that is substantially inhibited by a serineprotease inhibitor. Such inhibition can be measured by techniques knownto those skilled in the art. To be substantially inhibited means that atleast half of the proteolytic activity of the protease protein isinhibited by a serine protease inhibitor. Preferably at least about 70percent, and even more preferably at least about 90 percent of theproteolytic activity of the protease protein is inhibited by a serineprotease inhibitor.

An isolated protein of the present invention can be produced in avariety of ways, including recovering such a protein from a flea midgutand producing such a protein recombinantly. In one embodiment, a fleamidgut protease can be recovered by methods heretofore disclosed forobtaining a soluble flea midgut preparation. A flea midgut proteaseprotein can be further purified from a disrupted flea midgut by a numberof techniques known to those skilled in the art, including, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis (e.g., standard, capillary and flow-throughelectrophoresis), hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization. In oneembodiment, a flea midgut protease is purified using protease inhibitoraffinity chromatography, an example of which is disclosed in theExamples section.

Another embodiment of the present invention is a method to produce anisolated protein of the present invention using recombinant DNAtechnology. Such a method includes the steps of (a) culturing arecombinant cell comprising a nucleic acid molecule encoding a proteinof the present invention to produce the protein and (b) recovering theprotein therefrom. Details on producing recombinant cells and culturingthereof are presented below. The phrase "recovering the protein" referssimply to collecting the whole fermentation medium containing theprotein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, as heretoforedisclosed.

Isolated proteins of the present invention are preferably retrieved in"substantially pure" form. As used herein, "substantially pure" refersto a purity that allows for the effective use of the protein as avaccine. A vaccine for animals, for example, should exhibit nosubstantial toxicity and should be capable of stimulating the productionof antibodies in a vaccinated animal.

Another embodiment of the present invention is an isolated nucleic acidmolecule capable of hybridizing under stringent conditions with a geneencoding a flea protease present in a flea midgut. Such a nucleic acidmolecule is also referred to herein as a flea protease nucleic acidmolecule. Particularly preferred is an isolated nucleic acid moleculethat hybridizes under stringent conditions with a flea serine proteasegene or with a flea aminopeptidase gene. The characteristics of suchgenes are disclosed herein. In accordance with the present invention, anisolated nucleic acid molecule is a nucleic acid molecule that has beenremoved from its natural milieu (i.e., that has been subject to humanmanipulation). As such, "isolated" does not reflect the extent to whichthe nucleic acid molecule has been purified. An isolated nucleic acidmolecule can include DNA, RNA, or derivatives of either DNA or RNA.

As stated above, a flea protease gene includes all nucleic acidsequences related to a natural flea protease gene such as regulatoryregions that control production of a flea protease protein encoded bythat gene (such as, but not limited to, transcription, translation orpost-translation control regions) as well as the coding region itself. Anucleic acid molecule of the present invention can be an isolatednatural flea protease nucleic acid molecule or a homologue thereof. Anucleic acid molecule of the present invention can include one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof. The minimal size of a flea protease nucleic acidmolecule of the present invention is the minimal size capable of forminga stable hybrid under stringent hybridization conditions with acorresponding natural gene. Flea protease nucleic acid molecules canalso include a nucleic acid molecule encoding a hybrid protein, a fusionprotein, a multivalent protein or a truncation fragment.

An isolated nucleic acid molecule of the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. As used herein, the phrase "at least a portion of" an entityrefers to an amount of the entity that is at least sufficient to havethe functional aspects of that entity. For example, at least a portionof a nucleic acid sequence, as used herein, is an amount of a nucleicacid sequence capable of forming a stable hybrid with the correspondinggene under stringent hybridization conditions.

An isolated nucleic acid molecule of the present invention can also beproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatedflea protease nucleic acid molecules include natural nucleic acidmolecules and homologues thereof, including, but not limited to, naturalallelic variants and modified nucleic acid molecules in whichnucleotides have been inserted, deleted, substituted, and/or inverted insuch a manner that such modifications do not substantially interferewith the nucleic acid molecule's ability to encode a flea proteaseprotein of the present invention or to form stable hybrids understringent conditions with natural nucleic acid molecule isolates.

A flea protease nucleic acid molecule homologue can be produced using anumber of methods known to those skilled in the art (see, for example,Sambrook et al., ibid.). For example, nucleic acid molecules can bemodified using a variety of techniques including, but not limited to,classic mutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures and ligation ofmixture groups to "build" a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule homologues can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid (e.g., the ability of ahomologue to elicit an immune response against a flea protease and/or tohave proteolytic activity) and/or by hybridization with isolated fleaprotease nucleic acids under stringent conditions.

An isolated flea protease nucleic acid molecule of the present inventioncan include a nucleic acid sequence that encodes at least one fleaprotease protein of the present invention, examples of such proteinsbeing disclosed herein. Although the phrase "nucleic acid molecule"primarily refers to the physical nucleic acid molecule and the phrase"nucleic acid sequence" primarily refers to the sequence of nucleotideson the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding an flea proteaseprotein.

One embodiment of the present invention is a flea protease nucleic acidmolecule of the present invention that is capable of hybridizing understringent conditions to a nucleic acid that encodes at least a portionof a flea protease or a homologue thereof. Preferred is a flea proteasenucleic acid molecule that includes a nucleic acid sequence having atleast about 65 percent, preferably at least about 75 percent, morepreferably at least about 85 percent, and even more preferably at leastabout 95 percent homology with the corresponding region(s) of thenucleic acid sequence encoding at least a portion of a flea proteaseprotein. Particularly preferred is a flea protease nucleic acid moleculecapable of encoding at least a portion of a flea protease that naturallyis present in flea midguts and preferably is included in a soluble fleamidgut preparation of the present invention. Examples of nucleic acidmolecules of the present invention are disclosed in the Examplessection.

A preferred flea serine protease nucleic acid molecule of the presentinvention is a nucleic acid molecule that hybridizes under stringenthybridization conditions with at least one of the following nucleic acidmolecules: nfSP1, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8,nfSP9, nfSP10, nfSP11, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16 and/ornfSP17. More preferred is a nucleic acid molecule that hybridizes understringent hybridization conditions with at least one of the followingnucleic acid molecules: nfSP4₆₇₂, nfSP1₁₅₆, nfSP2₁₆₈, nfSP3₁₇₇,nfSP4₁₅₆, nfSP5₁₅₉, nfSP6₁₆₈, nfSP7₁₅₉, nfSP8₁₈₆, nfSP9₁₆₈, nfSP10₁₂₀,and/or nfSP11₁₆₂. Even more preferred are nucleic acid molecules thatinclude nfSP1, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8, nfSP9,nfSP10, nfSP11, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16 and/or nfSP17,and even more particularly, nfSP4₆₇₂, nfSP1₁₅₆, nfSP2₁₆₈, nfSP3₁₇₇,nfSP4₁₅₆, nfSP5₁₅₉, nfSP6₁₆₈, nfSP7₁₅₉, nfSP8₁₈₆, nfSP9₁₆₈, nfSP10₁₂₀,and/or nfSP11₁₆₂.

Particularly preferred flea serine protease nucleic acid moleculesinclude at least one of the following sequences: SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, and/or nucleic acid sequences disclosed in Table 2. Alsopreferred are allelic variants of such nucleic acid molecules.

A preferred flea aminopeptidase nucleic acid molecule of the presentinvention is a nucleic acid molecule that hybridizes under stringenthybridization conditions with nfAP₄₅₃. More preferred is anaminopeptidase nucleic acid molecule that includes nfAP₄₅₃. Particularlypreferred is a nucleic acid molecule that includes nucleic acid sequenceSEQ ID NO:50 or an allelic variant thereof.

Knowing a nucleic acid molecule of a flea protease protein of thepresent invention allows one skilled in the art to make copies of thatnucleic acid molecule as well as to obtain a nucleic acid moleculeincluding additional portions of flea protease protein-encoding genes(e.g., nucleic acid molecules that include the translation start siteand/or transcription and/or translation control regions), and/or fleaprotease nucleic acid molecule homologues. Knowing a portion of an aminoacid sequence of a flea protease protein of the present invention allowsone skilled in the art to clone nucleic acid sequences encoding such aflea protease protein. In addition, a desired flea protease nucleic acidmolecule can be obtained in a variety of ways including screeningappropriate expression libraries with antibodies which bind to fleaprotease proteins of the present invention; traditional cloningtechniques using oligonucleotide probes of the present invention toscreen appropriate libraries or DNA; and PCR amplification ofappropriate libraries, or RNA or DNA using oligonucleotide primers ofthe present invention (genomic and/or cDNA libraries can be used). Toisolate flea protease nucleic acid molecules, preferred cDNA librariesinclude cDNA libraries made from unfed whole fleas, fed whole fleas, fedflea midguts, unfed flea midguts, and flea salivary glands. Techniquesto clone and amplify genes are disclosed, for example, in Sambrook etal., ibid. The Examples section includes examples of the isolation ofcDNA sequences encoding flea protease proteins of the present invention.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of aflea protease protein. oligonucleotides of the present invention can beRNA, DNA, or derivatives of either. The minimal size of sucholigonucleotides is the size required to form a stable hybrid between agiven oligonucleotide and the complementary sequence on another nucleicacid molecule of the present invention. Minimal size characteristics aredisclosed herein. The size of the oligonucleotide must also besufficient for the use of the oligonucleotide in accordance with thepresent invention. Oligonucleotides of the present invention can be usedin a variety of applications including, but not limited to, as probes toidentify additional nucleic acid molecules, as primers to amplify orextend nucleic acid molecules or in therapeutic applications to inhibitflea protease production. Such therapeutic applications include the useof such oligonucleotides in, for example, antisense-, triplexformation-, ribozyme- and/or RNA drug-based technologies. The presentinvention, therefore, includes such oligonucleotides and methods tointerfere with the production of flea protease proteins by use of one ormore of such technologies.

The present invention also includes a recombinant vector, which includesa flea protease nucleic acid molecule of the present invention insertedinto any vector capable of delivering the nucleic acid molecule into ahost cell. Such a vector contains heterologous nucleic acid sequences,that is nucleic acid sequences that are not naturally found adjacent toflea protease nucleic acid molecules of the present invention. Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid. Recombinant vectors can be used inthe cloning, sequencing, and/or otherwise manipulating of flea proteasenucleic acid molecules of the present invention. One type of recombinantvector, herein referred to as a recombinant molecule and described inmore detail below, can be used in the expression of nucleic acidmolecules of the present invention. Preferred recombinant vectors arecapable of replicating in the transformed cell. Preferred nucleic acidmolecules to include in recombinant vectors of the present invention aredisclosed herein.

As heretofore disclosed, one embodiment of the present invention is amethod to produce a flea protease protein of the present invention byculturing a cell capable of expressing the protein under conditionseffective to produce the protein, and recovering the protein. Apreferred cell to culture is a recombinant cell that is capable ofexpressing the flea protease protein, the recombinant cell beingproduced by transforming a host cell with one or more nucleic acidmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a host cell are disclosed herein.

Suitable host cells to transform include any cell that can betransformed and that can express the introduced flea protease protein.Such cells are, therefore, capable of producing flea protease proteinsof the present invention after being transformed with at least onenucleic acid molecule of the present invention. Host cells can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Suitable host cells of the present inventioncan include bacterial, fungal (including yeast), insect, animal andplant cells. Preferred host cells include bacterial, yeast, insect andmammalian cells, with bacterial (e.g., E. coli) and insect (e.g.,Spodoptera) cells being particularly preferred.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence which is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminth, insect and mammalian cells, such as, butnot limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB,bacteriophage lambda (λ) (such as λp_(L) and λp_(R) and fusions thatinclude such promoters), bacteriophage T7, T7lac, bacteriophage T3,bacteriophage SP6, bacteriophage SP01, metallothionein, alpha matingfactor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such asSindbis virus subgenomic promoters), baculovirus, Heliothis zea insectvirus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus40, retrovirus actin, retroviral long terminal repeat, Rous sarcomavirus, heat shock, phosphate and nitrate transcription control sequencesas well as other sequences capable of controlling gene expression inprokaryotic or eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers aswell as lymphokine-inducible inducible promoters (e.g., promotersinducible by interferons or interleukins). Transcription controlsequences of the present invention can also include naturally occurringtranscription control sequences naturally associated with a DNA sequenceencoding a flea protease protein.

Expression vectors of the present invention may also contain secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed flea protease protein to be secreted from the cell thatproduces the protein. Suitable signal segments include a flea proteaseprotein signal segment or any heterologous signal segment capable ofdirecting the secretion of a flea protease protein, including fusionproteins, of the present invention. Preferred signal segments include,but are not limited to, flea protease, tissue plasminogen activator(t-PA), interferon, interleukin, growth hormone, histocompatibility andviral envelope glycoprotein signal segments.

Expression vectors of the present invention may also contain fusionsequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of a flea protease nucleic acid molecule of thepresent invention can enhance the stability during production, storageand/or use of the protein encoded by the nucleic acid molecule.Furthermore, a fusion segment can function as a tool to simplifypurification of a flea protease protein, such as to enable purificationof the resultant fusion protein using affinity chromatography. Asuitable fusion segment can be a domain of any size that has the desiredfunction (e.g., increased stability and/or purification tool). It iswithin the scope of the present invention to use one or more fusionsegments. Fusion segments can be joined to amino and/or carboxyl terminiof a flea protease protein. Linkages between fusion segments and fleaprotease proteins can be constructed to be susceptible to cleavage toenable straight-forward recovery of the flea protease proteins. Fusionproteins are preferably produced by culturing a recombinant celltransformed with a fusion nucleic acid sequence that encodes a proteinincluding the fusion segment attached to either the carboxyl and/oramino terminal end of a flea protease protein.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed. A preferred recombinantmolecule includes one or more nucleic acid molecules of the presentinvention, with those that encode one or more flea protease proteins,and particularly one or more flea serine protease and/or aminopeptidaseproteins, being more preferred. Similarly, a preferred recombinant cellincludes one or more nucleic acid molecules of the present invention,with those that encode one or more flea protease proteins, andparticularly one or more flea serine protease and/or aminopeptidaseproteins, being more preferred.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

In accordance with the present invention, recombinant cells can be usedto produce flea protease proteins of the present invention by culturingsuch cells under conditions effective to produce such a protein, andrecovering the protein. Effective conditions to produce a proteininclude, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate, or effective, medium refers to any medium in which a cellof the present invention, when cultured, is capable of producing a fleaprotease protein. Such a medium is typically an aqueous mediumcomprising assimilable carbohydrate, nitrogen and phosphate sources, aswell as appropriate salts, minerals, metals and other nutrients, such asvitamins. The medium may comprise complex nutrients or may be a definedminimal medium.

Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantflea protease proteins may either remain within the recombinant cell; besecreted into the fermentation medium; be secreted into a space betweentwo cellular membranes, such as the periplasmic space in E. coli; or beretained on the outer surface of a cell or viral membrane. Methods topurify such proteins are heretofore disclosed.

The present invention also includes isolated anti-flea proteaseantibodies and their use to reduce flea infestation on a host animal aswell as in the environment of the animal. An anti-flea protease antibodyis an antibody capable of selectively binding to a protease present in aflea midgut, including female and male fed midguts as well as female andmale unfed midguts. An anti-flea protease antibody preferably binds tothe protease in such a way as to reduce the proteolytic activity of thatprotease.

Isolated antibodies are antibodies that have been removed from theirnatural milieu. The term "isolated" does not refer to the state ofpurity of such antibodies. As such, isolated antibodies can includeanti-sera containing such antibodies, or antibodies that have beenpurified to varying degrees. As used herein, the term "selectively bindsto" refers to the ability of such antibodies to preferentially bind tothe protease against which the antibody was raised (i.e., to be able todistinguish that protease from unrelated components in a mixture.).Binding affinities typically range from about 10³ M⁻¹ to about 10¹² M⁻¹.Binding can be measured using a variety of methods known to thoseskilled in the art including immunoblot assays, immunoprecipitationassays, radioimmunoassays, enzyme immunoassays (e.g., ELISA),immunofluorescent antibody assays and immunoelectron microscopy; see,for example, Sambrook et al., ibid.

Antibodies of the present invention can be either polyclonal ormonoclonal antibodies. Antibodies of the present invention includefunctional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein used to obtain the antibodies. Antibodies of the presentinvention also include chimeric antibodies that can bind to more thanone epitope. Preferred antibodies are raised in response to proteinsthat are encoded, at least in part, by a flea protease nucleic acidmolecule of the present invention.

Anti-flea antibodies of the present invention include antibodies raisedin an animal administered a flea protease vaccine of the presentinvention that exert their effect when fleas feed from the vaccinatedanimal's blood containing such antibodies. Anti-flea antibodies of thepresent invention also include antibodies raised in an animal againstone or more flea protease proteins, or soluble flea midgut preparations,of the present invention that are then recovered from the animal usingtechniques known to those skilled in the art. Yet additional antibodiesof the present invention are produced recombinantly using techniques asheretofore disclosed for flea protease proteins of the presentinvention. Antibodies produced against defined proteins can beadvantageous because such antibodies are not substantially contaminatedwith antibodies against other substances that might otherwise causeinterference in a diagnostic assay or side effects if used in atherapeutic composition.

Anti-flea protease antibodies of the present invention have a variety ofuses that are within the scope of the present invention. For example,such antibodies can be used in a composition of the present invention topassively immunize an animal in order to protect the animal from fleainfestation. Anti-flea antibodies can also be used as tools to screenexpression libraries and/or to recover desired proteins of the presentinvention from a mixture of proteins and other contaminants.Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to fleas in order to kill fleas. Targeting can beaccomplished by conjugating (i.e., stably joining) such antibodies tothe cytotoxic agents using techniques known to those skilled in the art.

A preferred anti-flea protease antibody of the present invention canselectively bind to, and preferentially reduce the proteolytic activityof, a flea serine protease, a flea metalloprotease, a flea aspartic acidprotease and/or a flea cysteine protease. More preferred anti-fleaprotease antibodies include anti-flea serine protease antibodies,anti-flea metalloprotease antibodies, and anti-flea aminopeptidaseantibodies. Particularly preferred are anti-flea serine proteaseantibodies and anti-flea aminopeptidase antibodies, including thoseraised against flea serine protease proteins or flea aminopeptidaseproteins of the present invention.

The present invention also includes the use of protease inhibitors thatreduce proteolytic activity of flea proteases to reduce flea infestationof animals and the surrounding environment. As used herein, proteaseinhibitors are compounds that interact directly with a protease therebyinhibiting that protease's activity, usually by binding to or otherwiseinteracting with the protease's active site. Protease inhibitors areusually relatively small compounds and as such differ from anti-proteaseantibodies that interact with the active site of a protease.

Protease inhibitors can be used directly as compounds in compositions ofthe present invention to treat animals as long as such compounds are notharmful to the animals being treated. Protease inhibitors can also beused to identify preferred types of flea proteases to target usingcompositions of the present invention. For example, the inventors haveshown herein the predominance of serine proteases in flea midguts,particularly in soluble flea midgut preparations, using proteaseinhibitors. Such knowledge suggests that effective reduction of fleainfestation of an animal can be achieved using serine protease vaccines,anti-flea serine protease antibodies and other inhibitors of serineprotease synthesis and activity that can be tolerated by the animal.That other proteases are also present in flea midguts according to thepresent invention also suggests targeting such proteases. Methods to useprotease inhibitors are known to those skilled in the art; examples ofsuch methods are disclosed herein.

In one embodiment, a protease inhibitor that can be used in acomposition of the present invention to treat an animal is identified bya method including the following steps: (a) identifying candidate (i.e.,putative, possible) inhibitor compounds by testing the efficacy of oneor more protease inhibitors (i) in vitro for their ability to inhibitflea protease activity and/or (ii) in a flea feeding assay for theirability to reduce the survival and/or fecundity of fleas by adding theinhibitors to the blood meal of a flea being maintained, for example, ina feeding system, such as that described by Wade et al., 1988, J.MedEntomol. 25, 186-190: and (b) testing the efficacy of the candidateinhibitor compounds in animals infested with fleas. Although one doesnot need both in vitro assay data and flea feeding assay data todetermine which candidate compounds to administer to animals, evaluationof both sets of data is preferred since data from neither of the assaysnecessarily predicts data to be obtained from the other assay. Forexample, candidate compounds identified using the in vitro assay maywork "in the test tube" but may not work in vivo for a number ofreasons, including the presence of interfering components in the bloodmeal that inhibit the activity of such compounds; e.g., althoughaprotinin can inhibit at least some flea serine proteases in vitro,aprotinin does not work well in the presence of serum proteins, such asare found in the blood. Furthermore, candidate inhibitor compoundsidentified by the flea feeding assays can include not only desiredcompounds but also compounds that reduce the viability and/or fecundityof fleas due to general toxicity (e.g., affecting the mitochondria offleas).

In another embodiment, protease inhibitors are used in the purificationof corresponding proteases by, for example, affinity chromatography, inwhich, a protease inhibitor is incubated with a mixture containing adesired protease under conditions that the inhibitor forms a complexwith the protease. The protease can then be recovered from the complex.The protease inhibitor can be attached to a solid support and/or belabelled with, for example, a radioactive, fluorescent, or enzymatic tagthat can be used to detect and/or recover the complex.

Suitable protease inhibitors to use in accordance with the presentinvention include serine protease inhibitors, metalloproteaseinhibitors, aspartic acid protease inhibitors, cysteine proteaseinhibitors, and/or aminopeptidase inhibitors. Preferred proteaseinhibitors include serine protease inhibitors, metalloproteaseinhibitors and aminopeptidase inhibitors, particularly those that arebroad spectrum inhibitors. More preferred are broad spectrum serineprotease inhibitors.

There is a wide variety of protease inhibitors, as is known to oneskilled in the art. Examples include, but are not limited to, AEBSF,aprotinin, bestatin, chloromethyl ketones TLCK (Nα-p-tosyl-L-lysinechloromethyl ketone) and TPCK (N-tosyl-L-phenylalanine chloromethylketone), chymostatin, cystatin, 3'4-dichloroisocoumarin, E-64(trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane), EDTA(ethylenediaminetetraacetic acid), leupeptin, methyl ketones having avariety of leaving groups, oxidized L-leucinethiol, pepstatin,1,10-orthophenanthroline, phosphoramidon, soybean trypsin/chymotrypsininhibitor and soybean trypsin inhibitor. Preferred protease inhibitorsfor use in the present invention include AEBSF, bestatin, E-64leupeptin, pepstatin, 1,10-orthophenanthroline, phosphoramidon, TLCK andTPCK, with AEBSF (a broad spectrum serine protease inhibitor), bestatin(an inhibitor of leucine aminopeptidase) and 1,10-orthophenanthroline (abroad spectrum metalloprotease inhibitor) being particularly preferred.

Protease inhibitors can be produced using methods known to those skilledin the art. Protein- or peptide-based protease inhibitors, such ascystatin or small peptides comprising a protease substrate, can beproduced recombinantly and modified as necessary.

The present invention also includes the use of proteolytically activeflea protease proteins of the present invention to identify additionalprotease inhibitors, and preferably protease inhibitor compounds thatcan be included in a composition of the present invention to beadministered to animals. A method to identify a flea protease inhibitorincludes the steps of (a) contacting (e.g., combining, mixing) anisolated flea protease protein with a putative (i.e., candidate)inhibitory compound under conditions in which, in the absence of thecompound, the protein has proteolytic activity, and (b) determining ifthe putative inhibitory compound inhibits the proteolytic activity ofthe protein. Putative inhibitory compounds to screen include organicmolecules, antibodies (including functional equivalents thereof) andsubstrate analogs. Methods to determine protease activity are known tothose skilled in the art, as heretofore disclosed. Particularlypreferred for use in identifying inhibitors are flea serine proteaseproteins and flea aminopeptidase proteins of the present invention.

The present invention also includes a test kit to identify a compoundcapable of inhibiting flea protease activity. Such a test kit includesan isolated flea protease protein having proteolytic activity and ameans for determining the extent of inhibition of proteolytic activityin the presence of (i.e., effected by) a putative inhibitory compound.

The present invention also includes inhibitors isolated by such amethod, and/or test kit, and their use to inhibit any flea protease thatis susceptible to such an inhibitor.

It is to be appreciated that the present invention also includesmimetopes of compounds of the present invention that can be used inaccordance with methods as disclosed for compounds of the presentinvention. As used herein, a mimetope of a proteinaceous compound of thepresent invention (e.g., a flea protease protein, an anti-flea proteaseantibody, a proteinaceous inhibitor of protease activity or synthesis)refers to any compound that is able to mimic the activity of thatproteinaceous compound, often because the mimetope has a structure thatmimics the proteinaceous compound. For example, a mimetope of a fleaprotease protein is a compound that has an activity similar to that ofan isolated flea protease protein of the present invention. Mimetopescan be, but are not limited to: peptides that have been modified todecrease their susceptibility to degradation; anti-idiotypic and/orcatalytic antibodies, or fragments thereof; non-proteinaceousimmunogenic portions of an isolated protein (e.g., carbohydratestructures); and synthetic or natural organic molecules, includingnucleic acids. Such mimetopes can be designed using computer-generatedstructures of proteins of the present invention. Mimetopes can also beobtained by generating random samples of molecules, such asoligonucleotides, peptides or other organic molecules, and screeningsuch samples by affinity chromatography techniques using thecorresponding binding partner.

The present invention includes therapeutic compositions, also referredto herein as compositions, that include a (i.e., at least one) compoundof the present invention. Preferred compounds to include in acomposition of the present invention include flea protease vaccines,anti-flea protease antibodies and/or protease inhibitors as disclosedherein. Such a therapeutic composition can protect an animal from fleainfestation by reducing flea protease activity, thereby reducing fleaburden on the animal and in the environment of the animal.

Particularly preferred therapeutic compositions of the present inventioninclude at least one of the following compounds: an isolated flea serineprotease protein or a mimetope thereof; an isolated flea serine proteasenucleic acid molecule that hybridizes under stringent hybridizationconditions with a flea serine protease gene; an isolated antibody thatselectively binds to a flea serine protease protein; an inhibitor offlea serine protease activity identified by its ability to inhibit fleaserine protease activity; an isolated flea aminopeptidase protein or amimetope thereof; an isolated flea aminopeptidase nucleic acid moleculethat hybridizes under stringent hybridization conditions with a fleaaminopeptidase gene; an isolated antibody that selectively binds to aflea aminopeptidase protein; and an inhibitor of flea aminopeptidaseactivity identified by its ability to inhibit flea aminopeptidaseactivity.

Another embodiment of the present invention is a therapeutic compositionthat includes a first compound that reduces flea protease activity and asecond compound that reduces flea burden by a method other than byreducing flea protease activity. The present invention also includes amethod to protect an animal from flea infestation by administering tothe animal such a composition. The first compound of such a compositionby effectively reducing flea protease activity in the midgut, enhancesthe activity of the second compound. While not being bound by theory, itis believed that a number of anti-flea treatments, particularly thosethat are proteinaceous, are not very effective because they are degradedin the flea midgut. The present invention permits the effective use ofsuch anti-flea treatments by reducing proteolytic degradation of suchtreatments by the flea midgut.

Preferred first compounds to include in such a composition include fleaprotease vaccines, anti-flea protease antibodies and/or proteaseinhibitors as disclosed herein.

Suitable second compounds include any anti-flea agent(s), including, butnot limited to, proteinaceous compounds, insecticides and flea collars.Preferred second compounds are proteinaceous compounds that effectactive immunization (e.g., antigen vaccines), passive immunization(e.g., antibodies), or that otherwise inhibit a flea activity that wheninhibited can reduce flea burden on and around an animal. Examples ofsecond compounds include a compound that inhibits binding between a fleamembrane protein and its ligand (e.g., a compound that inhibits fleaATPase activity or a compound that inhibits binding of a peptide orsteroid hormone to its receptor), a compound that inhibits hormone(including peptide or steroid hormones) synthesis, a compound thatinhibits vitellogenesis (including production of vitellin and transportand maturation thereof into a major egg yolk protein), a compound thatinhibits fat body function, a compound that inhibits flea muscle action,a compound that inhibits the flea nervous system, a compound thatinhibits the flea immune system and/or a compound that inhibits fleafeeding.

Compositions of the present invention can also include other componentssuch as a pharmaceutically acceptable excipient, an adjuvant, and/or acarrier. For example, compositions of the present invention can beformulated in an excipient that the animal to be treated can tolerate.Examples of such excipients include water, saline, Ringer's solution,dextrose solution, Hank's solution, and other aqueous physiologicallybalanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesameoil, ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In one embodiment of the present invention, the composition can alsoinclude an immunopotentiator, such as an adjuvant or a carrier.Adjuvants are typically substances that generally enhance the immuneresponse of an animal to a specific antigen. Suitable adjuvants include,but are not limited to, Freund's adjuvant; other bacterial cell wallcomponents; aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins; viral coat proteins; otherbacterial-derived preparations; gamma interferon; block copolymeradjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated animal. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, viruses, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to reduce protease activityin fleas feeding from the animal over a period of time ranging fromabout 1 to about 12 months. A controlled release formulation of thepresent invention is capable of effecting a treatment for preferably atleast about 1 month, more preferably at least about 3 months and evenmore preferably for at least about 6 months, even more preferably for atleast about 9 months, and even more preferably for at least about 12months.

In order to protect an animal from flea infestation, a therapeuticcomposition of the present invention is administered to the animal in aneffective manner such that the protease activity of fleas feeding fromthe blood stream of animals treated with the composition is reduced. Assuch, a treated animal is an animal that is competent to reduce the fleaburden by reducing flea protease activity, or by reducing flea proteaseactivity and at least one other flea activity. Preferably, the proteaseactivity is reduced by at least about 50 percent, more preferably by atleast about 70 percent and even more preferably by at least about 90percent. Methods to administer compositions to the animal in order torender the animal competent depend on the nature of the composition andadministration regime.

Animals administered a protease vaccine with at least one booster shotusually become competent at about the same time as would be expected forany vaccine treatment. For example, animals administered a booster doseabout 4 to 6 weeks after a primary dose usually become competent withinanother about 3 to 4 weeks. Animals administered a composition includingan anti-flea protease antibody or protease inhibitor become competent assoon as appropriate serum levels of the compound are achieved, usuallywith one to three days.

In a preferred embodiment, a composition of the present invention whenadministered to a host animal is able to reduce flea viability by atleast about 50 percent within at least about 21 days after the fleasbegin feeding from the treated animal. (Note that fleas usually liveabout 40 days to about 50 days on one or more animals.) A more preferredcomposition when administered to a host animal is able to reduce fleaviability by at least about 65 percent within at least about 14 daysafter the fleas begin feeding from the treated animal. An even morepreferred composition when administered to an animal is able to reduceflea viability by at least about 90 percent within at least about 7 daysafter the fleas begin feeding from the treated animal.

In another preferred embodiment, a composition of the present inventionwhen administered to a host animal is able to reduce flea fecundity(i.e., egg laying ability) by at least about 50 percent, more preferablyby at least about 70 percent, and even more preferably by at least about90 percent, within at least about 30 days after the fleas begin feedingfrom the treated animal. (Note that fleas usually do not begin layingeggs until about 7 days after taking a blood meal.)

In accordance with the present invention, compositions are administeredto an animal in a manner such that the animal becomes competent toreduce flea protease activity in a flea that feeds from the competent;i.e., the animal becomes a treated animal. For example, a flea proteasevaccine of the present invention, when administered to an animal in aneffective manner, is able to elicit (i.e., stimulate) an immune responsethat produces an antibody titer in the blood stream of the animalsufficient to reduce flea protease activity. Similarly, an anti-fleaprotease antibody of the present invention, when administered to ananimal in an effective manner, is administered in an amount so as to bepresent in the animal's blood stream at a titer that is sufficient toreduce flea protease activity. A protease inhibitor compound of thepresent invention, when administered to an animal in an effectivemanner, is administered in a manner so as to be present in the animal'sblood stream at a concentration that is sufficient to reduce fleaprotease activity. Oligonucleotide nucleic acid molecules of the presentinvention can also be administered in an effective manner, therebyreducing expression of flea proteases.

Compositions of the present invention can be administered to animalsprior to or during flea infestation. It is to be noted that whenvaccines of the present invention are administered to an animal, a timeperiod is required for the animal to elicit an immune response beforethe animal is competent to inhibit protease activity of fleas feedingfrom that animal. Methods to obtain an immune response in an animal areknown to those skilled in the art.

Acceptable protocols to administer compositions in an effective mannerinclude individual dose size, number of doses, frequency of doseadministration, and mode of administration. Determination of suchprotocols can be accomplished by those skilled in the art. A suitablesingle dose is a dose that is capable of protecting an animal from fleainfestation when administered one or more times over a suitable timeperiod. For example, a preferred single dose of a protease vaccine or amimetope thereof ranges from about 1 microgram (μg, also denoted ug) toabout 10 milligrams (mg) of the composition per kilogram body weight ofthe animal. Booster vaccinations can be administered from about 2 weeksto several years after the original administration. Booster vaccinationspreferably are administered when the immune response of the animalbecomes insufficient to protect the animal from flea infestation. Apreferred administration schedule is one in which from about 10 μg toabout 1 mg of the vaccine per kg body weight of the animal isadministered from about one to about two times over a time period offrom about 2 weeks to about 12 months. In one embodiment, a booster doseof a composition of the present invention is administered about 4 to 6weeks after the primary dose, and additional boosters are administeredabout once or twice a year. Modes of administration can include, but arenot limited to, oral, nasal, topical, transdermal, rectal, andparenteral routes. Parenteral routes can include, but are not limited tosubcutaneous, intradermal, intravenous, and intramuscular routes.

In another embodiment, a preferred single dose of an anti-flea proteaseantibody composition or a mimetope thereof ranges from about 1 μg toabout 10 mg of the composition per kilogram body weight of the animal.Anti-flea antibodies can be re-administered from about 1 hour to aboutbiweekly for several weeks following the original administration.Booster treatments preferably are administered when the titer ofantibodies of the animal becomes insufficient to protect the animal fromflea infestation. A preferred administration schedule is one in whichfrom about 10 μg to about 1 mg of an anti-flea protease antibodycomposition per kg body weight of the animal is administered about every2 to every 4 weeks. Suitable modes of administration are as disclosedherein and are known to those skilled in the art.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein(e.g., flea protease vaccine, anti-flea protease antibody, orproteinaceous protease inhibitor) or protective RNA (e.g., antisenseRNA, ribozyme or RNA drug) in the animal to be protected from disease.Nucleic acid molecules can be delivered to an animal in a variety ofmethods including, but not limited to, (a) direct injection (e.g., as"naked" DNA or RNA molecules, such as is taught, for example in Wolff etal., 1990, Science 247, 1465-1468) or (b) packaged as a recombinantvirus particle vaccine or as a recombinant cell vaccine (i.e., deliveredto a cell by a vehicle selected from the group consisting of arecombinant virus particle vaccine and a recombinant cell vaccine).

A recombinant virus particle vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging-deficient. A number ofrecombinant virus particles can be used, including, but not limited to,those based on alphaviruses, poxviruses, adenoviruses, herpesviruses,and retroviruses.

When administered to an animal, a recombinant virus particle vaccine ofthe present invention infects cells within the immunized animal anddirects the production of a protective protein or RNA nucleic acidmolecule that is capable of protecting the animal from disease caused bya parasite of the present invention. A preferred single dose of arecombinant virus particle vaccine of the present invention is fromabout 1×10⁴ to about 1×10⁷ virus plaque forming units (pfu) per kilogrambody weight of the animal. Administration protocols are similar to thosedescribed herein for protein-based vaccines.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells include Salmonella, E.coli, Mycobacterium, S. frugiperda, baby hamster kidney, myoblast G8,COS, MDCK and CRFK recombinant cells, with Salmonella recombinant cellsbeing more preferred. Such recombinant cells can be administered in avariety of ways but have the advantage that they can be administeredorally, preferably at doses ranging from about 10⁸ to about 10¹²bacteria per kilogram body weight. Administration protocols are similarto those described herein for protein-based vaccines. Recombinant cellvaccines can comprise whole cells or cell lysates.

Compositions of the present invention can be administered to any animalsusceptible to flea infestation, including warm-blooded animals.Preferred animals to treat include mammals and birds, with cats, dogs,humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits,raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail andturkeys as well as other furry animals, pets and/or economic foodanimals, being more preferred. Particularly preferred animals to protectare cats and dogs.

The present invention includes compositions to treat flea infestation byany flea. As such, compositions of the present invention can be derivedfrom any flea species. Preferred fleas to target include fleas of thefollowing genera: Ctenocephalides, Cyopsyllus, Diamanus (Oropsylla),Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla, with those ofthe species Ctenocephalides canis, Ctenocephalides felis, Diamanusmontanus, Echidnophaga gallinacea, Nosopsyllus faciatus, Pulex irritans,Pulex simulans, Tunga penetrans and Xenopsylla cheopis being morepreferred. Particularly preferred fleas from which to protect animalsinclude fleas of the species Ctenocephalides felis, Ctenocephalidescanis, and Pulex species (e.g., Pulex irritans and Pulex simulans). Itis also within the scope of the present invention to administercompositions of the present invention directly to fleas.

The present invention also includes the use of compositions of thepresent invention to reduce infestation by other ectoparasites as wellas the use of compositions including protease vaccines, anti-proteaseantibodies and compounds that inhibit protease synthesis and/or activityderived from any ectoparasite to reduce ectoparasite infestation,particularly controlled release formulations containing suchcompositions. Preferred ectoparasites to target include arachnids,insects and leeches. More preferred ectoparasites to target includefleas; ticks, including both hard ticks of the family Ixodidae (e.g.,Ixodes and Amblyomma) and soft ticks of the family Argasidae (e.g.,Ornithodoros, such as O. parkeri and O. turicata); flies, such as midges(e.g., Culicoides), mosquitos, sand flies, black flies, horse flies,horn flies, deer flies, tsetse flies, stable flies, myiasis-causingflies and biting gnats; ants; spiders, lice; mites; and true bugs, suchas bed bugs and kissing bugs, including those carrying Chagas disease.Even more preferred ectoparasites to target include fleas, mosquitos,midges, sandflies, blackflies, ticks and Rhodnius.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This Example demonstrates that soluble flea midgut preparations containserine protease activity as well as leucine aminopeptidase activity.

Using a homogenization/sonication protocol as described in U.S. Ser. No.07/806,482 now U.S. Pat. No. 5,356,622, ibid., followed by an about 2minute centrifugation step at about 10,000×g, soluble flea midgutpreparations were obtained from fed and unfed fleas. Pellets from thecentrifugation step were also collected and resuspended for analysis.Also prepared were whole flea lysates. Peptide substrate screeningstudies using the synthetic chromogenic trypsin substrate BAPNA(Nα-benzoyl-DL-arginine-p-nitroanilide; available from Sigma ChemicalCo., St. Louis Mo.) demonstrated significant proteolytic activity inboth soluble flea midgut preparations as well as some proteolyticactivity in resuspended midgut pellets. Soluble unfed flea midgutpreparations exhibited about 10 times as much activity as did controls(samples to which no flea midgut fractions were added), whereas solublefed flea midgut preparations exhibited about 20 times as much activityas did controls. Whole flea preparations exhibited about 2 to 3 times asmuch activity as did controls.

The ability of soluble fed and unfed flea midgut preparations to cleaveBAPNA was almost completely inhibited (i.e., nearly 100%) by aprotinin(available from Sigma), whereas PMSF (phenylmethane-7-sulfonyl fluoride;available from Sigma) inhibited such proteolytic activity by about 50%.EDTA inhibited proteolytic activity of the preparations by about 10%,whereas addition of calcium ions stimulated proteolytic activity byabout 25%. These results indicate the presence of serine proteaseactivity, and more particularly of trypsin-like activity, in thesesoluble flea midgut preparations. These results also suggest thepresence of serine protease isoforms in the preparations. It is also ofinterest to note that flea trypsin-like activity appears to bedistinctive from that of mosquitos in that mosquito trypsins are notaffected by EDTA or calcium ions.

Using a methyl-hemoglobin substrate, the pH optimum of the proteolyticactivity in the soluble flea midgut preparations was found to be betweenpH 7 and pH 9, with a pH of about pH 8 giving the best activity. Such pHoptima suggest the presence of serine proteases in soluble flea midgutpreparations.

Soluble preparations of both unfed and fed flea midgut solublepreparations also were able to cleave the leucine aminopeptidasespecific substrate LPNA (L-leucine-p-nitroanilide; available from Sigma)using standard conditions, indicating the presence of leucineaminopeptidase (LAP) activity in such preparations.

Example 2

The following example evaluated the number of proteases in flea midgutsthat could be assessed by protease substrate gel analysis.

Protease substrate gels (available from Novex, San Diego, Calif., asNovex Zymogels) were 10% polyacrylamide-SDS gels with 0.1% gelatin.Samples and gels were processed according to Novex instructions.Briefly, samples were diluted in SDS-PAGE sample buffer without reducingagents. Tris-glycine SDS-PAGE was carried out by standard procedures.After electrophoresis, gels were incubated in 0.25% Triton X-100 at roomtemperature for 30 minutes (min), then in developing buffer (50 mM(millimolar) Tris-HCl pH 7.0, 5 mM CaCl₂, 0.02% Brij 35, 0.2 M (molar)NaCl) at room temperature for 30 min, and then incubated with freshdeveloping buffer at 37° C., usually overnight. Gels were then stained30 min in 0.5% coomassie R-250, 40% methanol, 10% acetic acid anddestained in 40% methanol, 10% acetic acid.

The following flea midguts were dissected directly into sample buffer:100 midguts from unfed males; 100 midguts from unfed females; 100midguts from fed males; and 100 midguts from fed females. Samplescontaining 10 or 20 midguts each were evaluated using protease substrategel analysis and numerous negative staining bands were observed. Thegeneral pattern was the same for female and male midguts, although thereappeared to be more activity in gel lanes containing female midguts.There were distinct differences noted between gel lanes containing fedand unfed midguts. There was a definite increase in overall activity inthe fed midgut lanes, and, in addition, there were differences in theband patterns.

Fed and unfed female midguts were further evaluated using proteasesubstrate gel analysis and the results are shown in FIG. 1. The proteasesubstrate gel shown in FIG. 1 demonstrates the relative proteolyticactivity in 1, 2, 5 or 10 midguts from either fed or unfed female fleas.Specifically, lane 1 contains a set of molecular weight markers. Lanes 2through 5 contain, respectively, 10, 5, 2 and 1 unfed midguts. Lanes 6through 9 contain, respectively, 1, 2, 5 and 10 fed midguts. Lane 10contains 100 μg of dried bovine blood.

Proteolytic activity could easily be detected in one fed or one unfedfemale midgut, although there was considerably more activity in the fedmidgut. Lane 10 evaluated 100 μg of dried bovine blood to assess if theincrease in activity seen in the fed midgut lane was due to proteases inthe blood meal. No activity was seen in the blood lane.

Example 3

This example evaluated the protease classes present in flea midguts.

Three unfed female midguts and 0.75 fed female midguts were evaluated induplicate in several protease substrate gels. Each gel was cut in half.Half was processed as described in Example 2, while the other halfcontained protease inhibitors in all incubation buffers. The followinginhibitors were evaluated:

(a) the serine protease inhibitor AEBSF (available from BoehringerMannheim, Indianapolis, Ind.) was used at a final concentration of 1 mM;

(b) the serine protease inhibitor soybean trypsin inhibitor (availablefrom Sigma) was used at a final concentration of 100 μg/ml (milliliter);

(c) the cysteine and serine protease inhibitor leupeptin (available fromSigma) was used at a final concentration of 10 μg/ml;

(d) the aminopeptidase inhibitor bestatin (available from Sigma) wasused at a final concentration of 0.25 mM;

(e) the metalloprotease inhibitor EDTA (available from Sigma) was usedat a final concentration of 2 mM; and

(f) the cysteine protease E-64 (available from Sigma) was used at afinal concentration of 10 μg/ml.

AEBSF, soybean trypsin inhibitor and leupeptin were the only inhibitorsto have any effect at the sensitivity of this assay. It was determinedthat serine proteases were the predominant, if not only, proteasespresent in the midgut preparations evaluated. FIG. 2 shows a proteasesubstrate gel with fed (lanes 2 and 4) and unfed (lanes 1 and 3) midgutpreparations with (lanes 3 and 4) and without (lanes 1 and 2) AEBSF.Residual activity in the inhibitor lanes could have been due toproteolysis that occurred during electrophoresis and prior to saturationof the gel with inhibitor in the incubation buffers.

Example 4

This Example evaluates protease activity contained in a soluble fedmidgut preparation of the present invention.

Mixed-sex fed flea midguts were processed as described in U.S. Ser. No.07/806,482, ibid. Aliquots of several steps of the procedure wereevaluated by loading an equivalent of 0.4 midguts per lane of a proteasesubstrate gel as described in Example 2. The results are shown in FIG.3. Samples were from the low speed supernatant (lanes 2 and 9),sonicated midguts (lanes 3 and 10), high speed supernatant (lanes 4 and11), combined low and high speed supernatants (FGS) (lanes 5 and 12) andthe high speed pellet (lanes 6 and 13). Lanes 7 and 8 contained 50nanograms (ng) of trypsin as a control. Duplicate lanes were evaluated.The gel was cut in half, and lanes 1-7 were processed as described inExample 2, and lanes 8-14 were processed with 100 μg/ml soybean trypsininhibitor in all the incubation buffers.

Protease activity was seen in all preparations, the most being observedin the FGS lane (lane 5). It was also evident that the majority of theactivity was inhibited by soybean trypsin inhibitor, a serine proteaseinhibitor.

Example 5

This Example demonstrates the increase in flea midgut protease activityafter blood feeding by fleas.

Fleas were fed on a dog for 15 minutes. At timed intervals afterfeeding, two midguts were dissected directly into sample buffer andproteases evaluated by protease substrate gel analysis as described inExample 2. FIG. 4 depicts a gel showing midgut protease activity at 30min (lane 1), 1 hr (lane 2), 2 hr (lane 3), 4 hr (lane 4), 6 hr (lane5), 8 hr (lane 6), 24 hr (lane 7) and 56 hr (lane 8) after blood feedingended.

Increases in proteolytic activity were first observed 2 hr (lane 3)after feeding, although at 4 hr (lane 4) there was a much greaterincrease in activity noted. This increase in activity was still noticed56 hr after feeding (lane 8).

Example 6

This Example evaluates the effect of a number of protease inhibitors onflea viability and fecundity in a flea feeding system as described byWade et al. ibid.

The following protease inhibitors were tested at the indicated finalconcentrations in blood meals:

(a) Aminopeptidase inhibitor bestatin at 1.3 mM and 13 mM;

(b) Aspartic acid protease inhibitor pepstatin A at 1 μg/ml and 10μg/ml;

(c) cysteine protease inhibitor E-64 at 1 μg/ml and 10 μg/ml.

(d) Metalloprotease inhibitor phosphoramidon at 10 μg/ml and 100 μg/ml;and

(e) the following serine protease inhibitors:

AEBSF at 0.3 mM, 0.5 mM, 5.0 mM and 6.0 mM;

Aprotinin at 2 μg/ml and 20 μg/ml;

Leupeptin at 5 μg/ml and 50 μg/ml;

Soybean trypsin inhibitor at 10 μg/ml and 100 μg/ml;

Soybean trypsin/chymotrypsin inhibitor at 10 μg/ml and 100 μg/ml;

AEBSF is available from Boehringer Mannheim; all other listed inhibitorsare available from Sigma.

Protease inhibitor compounds were tested in groups of 3 to 6 includingappropriate control groups. Inhibitors were not tested in groups ofcommon inhibition types. Rather, they were tested in groups based on thediluent needed to dissolve them. (AEBSF, aprotinin, bestatin, leupeptin,phosphoramidon, soybean trypsin inhibitor and soybeantrypsin/chymotrypsin inhibitor were dissolved in water; E-64 andpepstatin were dissolved in ethanol). This reduced the number of control(diluent only) groups needed within a particular assay. Inhibitorconcentrations were chosen such that the lower concentration used waswithin the range recommended by the supplier for that inhibitor. Thehigher concentration was typically 10 times above the lowerconcentration and was used to look for dose response.

The general protocol for all of the assays was as follows: Approximately2000 newly emerged adult fleas were placed in feeding chambers to feedon normal blood for about 24 to 48 hr. The fleas were prefed for tworeasons: The first was to be certain that only fleas that would feed inthe feeding system were used in the comparative study. The second was toprime female fleas for egg laying, since female fleas typically do notbegin laying maximal numbers of eggs per day until the third day offeeding.

The prefed fleas were placed in "minifeeder" feeding chambers at a ratioof about 80 female fleas to about 20 male fleas for a total of about 100fleas per chamber. Actual total number of fleas per chamber varied fromabout 90 to 125 fleas. Previous experiments have not demonstrated anydifferences in adult survival or fecundity based on such variance innumbers of fleas in a chamber. Three chambers were prepared for eachexperimental and control group. A fresh blood meal containing theappropriate inhibitor in 3 ml total volume was placed on each chamberdaily through the 7 day extent of an assay.

On days 3, 5, and 7 of the assay, surviving adult fleas were transferredto clean chambers. The contents of the original chambers were dissolvedin about 40 ml of PBS (phosphate-buffered saline) in a 50 ml Falcontube. The contents of a given tube were then filtered through apre-weighed #1 Whatman filter disk inserted into a vacuum filter. The 50ml tube and the filter funnel were rinsed with distilled water which wasthen passed through the filter. Once the chamber contents had beenfiltered, dead adult fleas were removed from the filter paper and placedin a labelled tube so that they could be counted and sexed. The filterpaper was then placed into a preweighed 12×75 polypropylene tube anddried in the SpeedVac for 2.5 hr with the heater on. After drying thefilter paper was weighed. The weight of the filter paper and tube wassubtracted to obtain the dry weight of the eggs and this value wasconverted to an estimated number of eggs using the formulay=41384.361x+162.37, where x=dry weight of eggs.

On day 7, adult fleas that had survived the study were frozen, countedand sexed. The numbers were added to the number of male and female fleasthat had died during the assay to verify the number of male and femalefleas in each chamber at the start of the study.

Female, male and total adult flea survival were calculated for allexperimental and control groups on days 3, 5, and 7 of each assay.Additionally, the number of eggs per surviving female was calculated ondays 3, 5 and 7. Female fleas found dead on a given collection date wereincluded in the total number of egg-laying females for the days betweenthat date and the previous collection date, providing a conservativeestimate of fecundity. Fecundity values were averaged for the threecollection dates to obtain an average for each group over 7 days.

Results of these studies are presented below in Table 1 and FIG. 6through FIG. 9. All survival and fecundity values are presented below asa percent of control value.

                  TABLE 1    ______________________________________    Effect of Protease Inhibitors    on Flea Viability and Fecundity                  Fecundity.sup.1                         Adult Survival.sup.1    Compound  Conc.     Days 1-7 Female                                       Male  Total    ______________________________________    AEBSF              6.0    mM     17.2%  4.1%  0.0   3.4%              5.0    mM     1.4%   6.8%  0.0%  5.6%              0.5    mM     95.0%  103.9%                                         104.2%                                               103.6%              0.3    mM     82.4%  116.2%                                         103.0%                                               111.9%    Aprotinin              20     ug/ml  84.2%  100.0%                                         101.7%                                               99.9%              2      ug/ml  83.2%  103.2%                                         104.9%                                               103.3%    Leupeptin              50     ug/ml  77.6%  101.5%                                         111.7%                                               104.6%              5      ug/ml  85.0%  71.0% 61.4% 68.4%    Soybean Trypsin    Inhibitor              100    ug/ml  79.1%  76.5% 76.0% 76.3%              10     ug/ml  96.1%  80.1% 101.7%                                               83.9%    Trypsin/    Chymotrypsin    Inhibitor              100    ug/ml  81.1%  88.0% 95.4% 89.9%              10     ug/ml  100.7% 115.1%                                         143.5%                                               120.7%    E-64              10     ug/ml  177.4% 110.2%                                         139.0%                                               114.2%              1      ug/ml  109.4% 99.9% 102.9%                                               100.1%              10     ug/ml  84.1%  90.2% 91.1% 90.6%              1      ug/ml  95.2%  77.3% 80.0% 77.5%    Phosphoramidon              100    ug/ml  84.9%  70.2% 64.6% 69.7%              10     ug/ml  89.0%  98.8% 95.2% 97.8%    Pepstatin A              10     ug/ml  83.9%  113.6%                                         133.4%                                               116.2%              1      ug/ml  67.7%  77.6% 96.6% 80.5%    Bestatin              13.0   mM     23.3%  121.0%                                         103.4%                                               117.0%              1.3    mM     60.4%  119.5%                                         116.3%                                               116.8%    ______________________________________     .sup.1 All experimental values are expressed as a percent of the     corresponding control group.

The aminopeptidase inhibitor bestatin caused a significant (p<0.05)reduction in fecundity at 13 mM (77% reduction) and at 1.3 mM (40%reduction) indicating the presence of an aminopeptidase or otherexopeptidase in flea midguts. Bestatin at the concentrations tested,however, had no significant effect on adult viability at eitherconcentration. These results suggest that aminopeptidases may play arole in ovarian function, or a related process, such as vitellogenesis.

The aspartic acid protease inhibitor pepstatin A caused a significantreduction (p<0.05) in fecundity at 1 μg/ml (32% reduction), but not at10 μg/ml. Pepstatin A had no significant effect on adult viability ateither concentration.

The cysteine protease inhibitor E-64 showed no statistically significantreduction in fecundity in this assay. There was a small, but significant(p<0.05), reduction in total adult flea survival when E-64 was dissolvedin grain alcohol and added to blood at 1 μg/ml. However, this reductionwas not evident in the group that was fed blood containing 10 μg/ml E-64in grain alcohol.

The metalloprotease inhibitor phosphoramidon caused a reduction in adultviability of about 30%, which, however was not statisticallysignificant. There was no significant reduction in fecundity.

Results using serine protease inhibitors were particularly interestingand suggest the significance of serine proteases in flea midguts. AEBSFadministered at concentrations ranging from about 5 mM to about 6 mMreduced flea fecundity by more than 80%. In addition, adult survival wasreduced to near zero (p<0.05).

Aprotinin, however, had no significant effect on either fecundity orviability, likely due to the ability of serum proteins, such as albumin,to interfere with aprotinin's inhibitory activity.

Leupeptin had no effect on fecundity at both concentrations, but reducedadult viability by 30% at 5 μg/ml. However, adult viability was notaffected by 50 μg/ml leupeptin and none of the observed reductions werestatistically significant.

Soybean trypsin inhibitor caused a small (20%) statisticallyinsignificant reduction in fecundity at 100 μg/ml. The lowerconcentration had no effect. Soybean trypsin inhibitor, on the otherhand, is very effective in in vitro studies as disclosed in several ofthe examples and was used to purify serine proteases as disclosed inExample 7. Soybean trypsin/chymotrypsin inhibitor had no effect on adultviability or fecundity.

Example 7

This Example describes the production of a preferred soluble flea midgutpreparation of the present invention and purification of flea serineproteases therefrom. Also included is amino acid sequence analysis of aflea serine protease of the present invention.

The soluble flea midgut preparation was prepared as follows. Fleamidguts (3,735) from a mix of female and male fed fleas were homogenizedin a homogenization buffer comprising 1.5 ml 50 mM Tris-HCl, 0.5 M NaCl,pH 8.5. The homogenate was centrifuged at 14,000×g for 10 min. Theresultant pellet was processed again in another 1.5 ml of thehomogenization. The two supernatant solutions were combined to form thesoluble flea midgut preparation.

The preparation was added to 3 ml of p-aminobenzamidine-sepharose 6 B(affinity matrix for trypsin-like proteases, available from Sigma) andincubated at 5° C. overnight on a rocker. The sepharose beads weredrained and washed with 7.5 ml of the homogenization buffer. Theadsorbed proteins were eluted with 5 ml 0.1 M p-aminobenzamidine in thesame buffer. This eluate was concentrated and the buffer exchanged to 50mM Tris-HCl pH 8.5, 0.1 mM CaCl₂ by ultrafiltration through a membranewith a 3 kD cutoff, the final volume being 140 μl (microliters).

Labeling of proteins was performed by adding 10 μl of (1,3-³H)-diisopropylfluorophosphate (available from New England Nuclear,Beverly, Mass., at 6.0 Ci (Curies)/mmole, 1.0 mCi/ml) to 90 μl of theaffinity purified proteins and incubating at 5° C. for 18 hours. Thereaction was divided in half, each half then being separated by C4reverse phase chromatography according to the following protocol:

Buffer A: 0.1% TFA in water

Buffer B: 0.085% TFA, 90% Acetonitrile

0.8 ml/min, 220 nm, 1 min fractions

5.6% B 15 min

5.6% to 100% B over 60 min

Ten microliters of each fraction was added to scintillation fluid andcounted. Most protein-associated counts were found in fractions 44-47.FIG. 5A shows electrophoresis of fractions 40 (lane 2), 44 (lane 3), 46(lane 4) and 47 (lane 5) from one chromatography run through a 14%Tris-glycine polyacrylamide-SDS gel, followed by coomassie staining.This gel was then processed with Entensigy (NEN) and exposed to film for18 hours, as shown in FIG. 5B. Each fraction contained several proteinsas shown in FIG. 5A, but only 4 bands were labeled, the most prominentbeing 26 kd (seen in lanes 3, 4 and 5), and denoted herein as PfSP26. Afaint band of 24 kd, denoted herein as PfSP24, was also noticed in lane5. A band of 19 kD, denoted herein as PfSP19, was labeled in lane 4 thatwas associated with a very faintly staining protein band. Some labeledproteins were seen at the dye front of lanes 4 and 5, indicating amolecular weight less than 6 kd, denoted herein as PfSP6, and could bedegradation products.

Fraction 44 (analogous to lane 3) from a second C4 chromatographyseparation experiment was electrophoresed, blotted onto PVDF, stainedwith Coomassie R-250 and destained via standard procedures. The 26 kdband, corresponding to PfSP26 (also referred to herein as PfSP44-E,indicating the fraction in which the protein eluted and the gel/filterband from which the protein was excised), was excised and subjected toN-terminal amino acid sequencing using techniques known to those skilledin the art. A partial N-terminal amino acid sequence of about 32 aminoacids was deduced and is represented herein as SEQ ID NO:1:

I I G G E V A G E G S A P Y Q V S L R T K E G N H F S G G S I L,

It should be noted that since amino acid sequencing technology is notentirely error-free, SEQ ID NO:1 represents, at best, an apparentpartial N-terminal amino acid sequence of PfSP26. This caution isparticularly relevant in light of the sequencing of this protein havingbeen done at a low picomolar concentration.

A homology search of the non-redundant protein sequence database wasperformed through the National Center for Biotechnology Informationusing the BLAST network. This database includes+SwissProt+PIR+SPUpdate+GenPept+GPUpdate.ole level. Results of thesearch indicate that the N-terminus of PfSP26 shares significant aminosequence homology with a number of serine proteases, including a varietyof trypsins, chymotrypsins and plasmins. The 32-amino acid N-terminalamino acid sequence of PfSP26 shared the highest degree of homology witha hornet chymotrypsin II.

Example 8

This example describes the cloning of certain flea protease nucleic acidmolecules of the present invention. This example also describes theproduction of certain recombinant molecules, recombinant cells and fleaprotease proteins of the present invention.

Several flea serine protease nucleic acid molecules, ranging in sizefrom about 250 to about 500 nucleotides, and representing one or morepartial flea serine protease genes, were PCR amplified from a fed fleamidgut cDNA library that was prepared from RNA isolated from fed fleamidguts using standard protocols as described in Sambrook et al., ibid.Several pairs of primers were used in PCR amplification reactions thatrepresented degenerate oligonucleotides designed from publishedsequences of serine protease genes isolated from biting insects (e.g.,mosquitos and black flies). Each primer pair was designed so that aproperly amplified fragment of a flea serine protease gene would includea domain corresponding to the most conserved domain of trypsin proteasegenes (thought to be the active site) given that such a domain iscontained in flea serine protease gene(s).

The amplified PCR fragments were of predicted size, ranging from about250 nucleotides to about 500 nucleotides, depending on which primerpairs were used. PCR fragments that hybridized to a probe designed fromthe domain most conserved among all known trypsin genes were gelpurified and cloned, for example, into the pCRII cloning vector(available from InVitrogen, Corp., San Diego, Calif.), followingmanufacturer's instructions. Nucleic acid sequences of the fragments arebeing determined using standard techniques.

The amplified PCR fragments are also being used as probes to identifyfull-length flea protease genes in unfed and fed flea midgut cDNAlibraries and in flea salivary gland cDNA libraries, as well as in fleagenomic DNA libraries, using standard procedures.

Recombinant molecules and recombinant cells including the amplified PCRfragments as well as full-length flea protease genes are being producedusing standard procedures. Culturing of such recombinant cells leads tothe production of flea protease proteins of the present invention.

Example 9

This Example describes the testing of a flea protease protein as a fleaprotease vaccine of the present invention, that is for the ability ofsuch a protein, upon administration to an animal, to elicit theproduction of antibodies that reduce flea protease activity and, assuch, reduce flea viability and/or fecundity. This Example alsodemonstrates the use of such a flea protease protein as a vaccine on adog subsequently infested with fleas.

A flea protease protein produced as described in Example 7 isadministered to rabbits according to a standard immunization protocolknown to those skilled in the art, including appropriate booster shots.Such a protein is also administered to guinea pigs and to dogs followinga similar protocol.

Sera is collected from the treated rabbits and is verified to containanti-flea protease antibodies. Such sera is then fed to fleas in afeeding system as reported by Wade et al. ibid. Fleas feeding on such asera show reduced viability compared to fleas feeding on sera collectedfrom rabbits not administered the flea protease protein. Sera fromtreated guinea pigs and dogs are verified in a similar manner.

Dogs treated with a flea protease protein are then infested with fleasas are dogs not treated with a flea protease protein. Dogs treated witha flea protease protein show a significant reduction in flea burdencompared to untreated dogs.

Example 10

This Example describes the determination of the partial N-terminal aminoacid sequence of additional flea serine protease proteins of the presentinvention.

An additional eight flea serine proteases were purified and consensuspartial N-terminal amino acid sequences were determined as described inExample 7. The results are as follows, the proteins being named by thefraction in which they were eluted and the SDS-PAGE gel band from whichthey were excised. Each of the proteases bore at least some sequencehomology to known proteases, the highest percent identity estimated tobe no more than about 30-40%.

Flea protease PfSP45-C had a partial N-terminal amino acid sequence of XV G G H D T S I D X H P H Q V T, also represented herein as SEQ ID NO:2.PfSP45-C was most similar in amino acid sequence to a fruit fly trypsinepsilon.

Flea protease PfSP46-C had a partial N-terminal amino acid sequence of IV G G A D A A P G N A P F Q V S L R D K G, also represented herein asSEQ ID NO:3. PfSP46-C was most similar in amino acid sequence to acollagenolytic 36 kD protease from a Kamchatda crab.

Flea protease PfSP46-A had a partial N-terminal amino acid sequence of IV G G Q D A D I A K Y G Y Q A S L Q V F N E H F X G A X I L N N Y, alsorepresented herein as SEQ ID NO:4. PfSP46-A was most similar in aminoacid sequence to a hornet chymotrypsin II.

Flea protease PfSP46-B had a partial N-terminal amino acid sequence of IV G G T D V N I E N F G W Q V S L F D R N G H F, also represented hereinas SEQ ID NO:5. PfSP46-B was most similar in amino acid sequence to afruit fly trypsin beta.

Flea protease PfSP48-A had a partial N-terminal amino acid sequence of IV G G H D T S I D K H P F Q V S L I D K N, also represented herein asSEQ ID NO:6. PfSP48-A was most similar in amino acid sequence to a fruitfly trypsin epsilon.

Flea protease PfSP48-B had a partial N-terminal amino acid sequence of VV G G L E A A E G S A P Y Q V X L Q W G N F, also represented herein asSEQ ID NO:7. PfSP48-B was most similar in amino acid sequence to a humanFactor 12.

Flea protease PfSP48-D had a partial N-terminal amino acid sequence of IV G G E D A E L G E X P T Q, also represented herein as SEQ ID NO:8.PfSP48-D was most similar in amino acid sequence to a bovine Factor 9.

Flea protease PfSP40-B had a partial N-terminal amino acid sequence of DE D G K D D S A P G E I, also represented herein as SEQ ID NO:9.PfSP40-B was most similar in amino acid sequence to a fruit flyfurin-like protease I.

Example 11

This Example describes the isolation of nucleic acid molecules encodingflea serine protease proteins of the present invention.

Several midgut proteinase cDNA genes have been isolated in a mannersimilar to that described in Example 8, using two degenerate primers,the design of which was based on a highly conserved serine proteinaseamino acid sequence (C Q/N G D S G G P L, denoted SEQ ID NO:10) locatedabout 195 amino acid residues (based on an average protease size ofabout 240 residues) from the mature amino terminus in a number of knownserine proteases. Complementing primers for use in the PCR amplificationreaction were primers corresponding to the vectors in which nucleic acidmolecules of the present invention had been ligated. The actual primersused in PCR amplification of serine protease nucleic acid molecules fromwhole fed flea cDNA expression libraries (produced as described inExample 8) included the following serine protease specific primers:cat-try #1 having nucleic acid sequence 5' TAA WGG WCC WCC YGA ATC TCCCTG GCA 3' (Y indicating C or T; W indicating A or T), representedherein as SEQ ID NO:11; and cat-try #2 having nucleic acid sequence 5'TAA WGG WCC AGA RTC TCC TTG ACA 3' (R indicating A or G), representedherein as SEQ ID NO:12. Vector specific primers included: M13 Reversehaving nucleic acid sequence 5' GGAAACAGCTATGACCATG 3', representedherein as SEQ ID NO:13; and T3 Primer having nucleic acid sequence 5'ATTAACCCTCACTAAAG 3', represented herein as SEQ ID NO:14. The resultantPCR products, obtained using standard PCR conditions (e.g., Sambrook etal., ibid.), were about 600 to about 700 nucleotides in length.

The PCR products were hybridized under standard hybridization conditions(e.g., Sambrook et al., ibid.) with (i.e., to) an internal syntheticoligonucleotide probe named H57, the sequence of which corresponds to aregion including a conserved histidine residue in known serineproteases. The nucleic acid sequence of H57 is 5' TGG GTW GTW ACW GCWGCW CAT TG 3', represented herein as SEQ ID NO:15. PCR products whichhybridized strongly to the probe were gel purified and cloned into theTA Vector™ (available from InVitrogen, Corp.). Approximately 80recombinant TA vector clones were isolated.

To prevent repetitive sequencing of the same serine proteinase clones, anumber of the clones were characterized to identify those having uniquerestriction endonuclease patterns using the enzymes HaeII and HaeIII.About 11 plasmids apparently containing unique flea serine proteinasenucleic acid molecules of about 600 to about 700 nucleotides in lengthwere isolated using this procedure. These nucleic acid molecules weresubjected to nucleic acid sequencing using the Sanger dideoxy chaintermination method, as described in Sambrook et al., ibid.

The complete nucleic acid sequence of one of the flea serine proteasenucleic molecules, namely nfSP4₆₇₂ is represented herein as SEQ IDNO:16. Translation of SEQ ID NO:16 yields a protein of about 223 aminoacids, denoted PfSP4₂₂₃, having amino acid sequence SEQ ID NO:17.Although the entire amino acid sequence of PfSP4₂₂₃ is not highlyconserved to that of known serine proteases, there are several conservedregions of note (as numbered for SEQ ID NO:17), including: (a) thesequence IVGG spanning from about amino acid 5 through about amino acid9; (b) the active-site histidine at about amino acid 46 and surroundingsequences spanning from about amino acid 41 through about amino acid 47;(c) the conserved aspartic acid residue at about amino acid 90; (d) theGWG sequence spanning from about amino acid 124 through about amino acid126; the conserved cysteines at about amino acid 152 and about aminoacid 165; and the conserved sequence around the active site serine,spanning from about amino acid 174 through about amino acid 182.

Nucleic acid and amino acid sequences of all 11 flea serine proteasenucleic acid molecules were determined for the regions corresponding tothe region in known serine proteases to span from the conserved GWGsequence to the conserved CXGDSGGP sequence (denoted SEQ ID NO:10). Fleanucleic acid molecule nfSP1₁₅₆ has the nucleic acid sequence representedherein as SEQ ID NO:18, which encodes a protein PfSP1₅₂ having an aminoacid sequence represented herein as SEQ ID NO:19. Flea nucleic acidmolecule nfSP2₁₆₈ has the nucleic acid sequence represented herein asSEQ ID NO:20, which encodes a protein PfSP2₅₆ having an amino acidsequence represented herein as SEQ ID NO:21. Flea nucleic acid moleculenfSP3₁₇₇ has the nucleic acid sequence represented herein as SEQ IDNO:22, which encodes a protein PfSP3₅₉ having an amino acid sequencerepresented herein as SEQ ID NO:23. Flea nucleic acid molecule nfSP4₁₅₆has the nucleic acid sequence represented herein as SEQ ID NO:24, whichencodes a protein PfSP4₅₂ having an amino acid sequence representedherein as SEQ ID NO:25. Flea nucleic acid molecule nfSP5₁₅₉ has thenucleic acid sequence represented herein as SEQ ID NO:26, which encodesa protein PfSP5₅₃ having an amino acid sequence represented herein asSEQ ID NO:27. Flea nucleic acid molecule nfSP6₁₆₈ has the nucleic acidsequence represented herein as SEQ ID NO:28, which encodes a proteinPfSP6₅₆ having an amino acid sequence represented herein as SEQ IDNO:29. Flea nucleic acid molecule nfSP7₁₅₉ has the nucleic acid sequencerepresented herein as SEQ ID NO:30, which encodes a protein PfSP7₅₃having an amino acid sequence represented herein as SEQ ID NO:31. Fleanucleic acid molecule nfSP8₁₈₆ has the nucleic acid sequence representedherein as SEQ ID NO:32, which encodes a protein PfSP8₆₂ having an aminoacid sequence represented herein as SEQ ID NO:33. Flea nucleic acidmolecule nfSP9₁₆₈ has the nucleic acid sequence represented herein asSEQ ID NO:34, which encodes a protein PfSP9₅₆ having an amino acidsequence represented herein as SEQ ID NO: 35. Flea nucleic acid moleculenfSP10₁₂₀ has the nucleic acid sequence represented herein as SEQ IDNO:36, which encodes a protein PfSP10₄₀ having an amino acid sequencerepresented herein as SEQ ID NO:37. Flea nucleic acid molecule nfSP11₁₆₂has the nucleic acid sequence represented herein as SEQ ID NO:38, whichencodes a protein PfSP11₅₄ having an amino acid sequence representedherein as SEQ ID NO:39.

Comparison of the nucleic acid sequences of the flea serine proteaseswith that of a mosquito (A. aegypti) trypsin indicates that SEQ ID NO:18is about 33% identical, SEQ ID NO:20 is about 33% identical, SEQ IDNO:22 is about 24% identical, SEQ ID NO:24 is about 25% identical, SEQID NO:26 is about 32% identical, SEQ ID NO:28 is about 38% identical,SEQ ID NO:30 is about 33% identical, SEQ ID NO:32 is about 33%identical, SEQ ID NO:34 is about 40% identical, SEQ ID NO:36 is about33% identical, and SEQ ID NO:38 is about 29% identical, to thecorresponding region of the mosquito trypsin. Comparison of the nucleicacid sequences of the flea serine proteases with that of a black fly (S.vittatum) trypsin indicates that SEQ ID NO:18 is about 34% identical,SEQ ID NO:20 is about 34% identical, SEQ ID NO:22 is about 25%identical, SEQ ID NO:24 is about 28% identical, SEQ ID NO:26 is about36% identical, SEQ ID NO:28 is about 45% identical, SEQ ID NO:30 isabout 29% identical, SEQ ID NO:32 is about 36% identical, SEQ ID NO:34is about 42% identical, SEQ ID NO:36 is about 34% identical, and SEQ IDNO:38 is about 30% identical, to the corresponding region of the blackfly trypsin. It is to be noted that the mosquito and black fly trypsinsare about 50% identical in the same regions.

Comparison of the amino acid sequences of the flea serine proteases withthat of a mosquito (A. aegypti) trypsin indicates that SEQ ID NO:19 isabout 11% identical, SEQ ID NO:21 is about 30% identical, SEQ ID NO:23is about 19% identical, SEQ ID NO:25 is about 19% identical, SEQ IDNO:27 is about 28% identical, SEQ ID NO:29 is about 21% identical, SEQID NO:31 is about 14% identical, SEQ ID NO:33 is about 22% identical,SEQ ID NO:35 is about 30% identical, SEQ ID NO:37 is about 22%identical, and SEQ ID NO:39 is about 29% identical, to the correspondingregion of the mosquito trypsin. Comparison of the amino acid sequencesof the flea serine proteases with that of a black fly (S. vittatum)trypsin indicates that SEQ ID NO:19 is about 14% identical, SEQ ID NO:21is about 28% identical, SEQ ID NO:23 is about 16% identical, SEQ IDNO:25 is about 17% identical, SEQ ID NO:27 is about 35% identical, SEQID NO:29 is about 33% identical, SEQ ID NO:31 is about 11% identical,SEQ ID NO:33 is about 22% identical, SEQ ID NO:35 is about 33%identical, SEQ ID NO:37 is about 21% identical, and SEQ ID NO:39 isabout 25% identical, to the corresponding region of the black flytrypsin. It is to be noted that the mosquito and black fly trypsins areabout 50% identical in the same regions.

Partial N-terminal amino acid sequences were deduced for each of thecloned flea serine protease nucleic acid molecules, four of which wereidentical to the following amino acid sequences derived from N-terminalsequencing of serine proteases as described in Example 10: SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:7. The remaining nucleic acidmolecules had the following deduced N-terminal amino acid sequences: SEQID NO:40, namely I V G G E N A K E K S D V P Y Q V S L R N A E N K H F CG G A I I D D Y W V L T, which was most similar in amino acid sequenceto mite fecal allergen Der pIII; SEQ ID NO:41, namely I V G G L E A K NG S A P F M V S L Q A E D Y F H, which was most similar in amino acidsequence to a chymotrypsin-like protein; SEQ ID NO:42, namely I I G G EV A G E G S A P Y Q V S L R T K E G N H F, which was most similar inamino acid sequence to a chymotrypsin-like protein; SEQ ID NO:43, namelyI V G G T A V D I R G F P G R Y Q F K P K P S F L W W F Y, which did notsubstantially match any protein in the data base; SEQ ID NO:44, namely IV N G L E A G V G Q F P I Q V F L D L T N I R D E K S R C G G A L F,which was most similar in amino acid sequence to a trypsin precursor;SEQ ID NO:45, namely I V G G L E A K N G I T P F I G F F A S G R L F,which was most similar in amino acid sequence to a chymotrypsin-likeprotease; SEQ ID NO:46, namely I V G G N D V S X K I F W Q V S I Q S N XQ H F C G, which was most similar in amino acid sequence to a trypsin;and SEQ ID NO:47, namely I I G G E D A P E G S A P Y Q V S L R N Q N L EH F C G G S I, which was most similar in amino acid sequence to achymotrypsin-like protein.

Additional amino terminal and carboxyl terminal sequences of flea serineprotease nucleic acid molecules comprising sequences listed above aswell as additional nucleic acid molecules identified using thetechniques described herein are presented in Table 2.

                                      TABLE 2    __________________________________________________________________________    Additional Flea Serine Protease Sequences    __________________________________________________________________________    A. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP1 (SEQ ID NOS:52 and 53) is:    TCA GCA CTC GTT GCC TTG TCT GCA GCT ATT CCT CAC TCC AAC AGA GTC     S   A   L   V   A   L   S   A   A   I   P   H   S   N   R   V    GTT GGA GGA CTG GAA GCT GCA GAG GGT TCT GCA CCT TAT CAA GTA TCC     V   G   G   L   E   A   A   E   G   S   A   P   Y   Q   V   S    TTG CAA GTT GGC AAC TTC CAC TTC TGT GGT GGT TCA ATT CTG AAC GAA     L   Q   V   G   N   F   H   F   C   G   G   S   I   L   N   E    TAT TGG GTT TTG ACT GCT GCT CAC TGT TTG GGT TAT GAC TTC GAC GTG     Y   W   V   L   T   A   A   H   C   L   G   Y   D   F   D   V    GTA GTT GGA ACA AAC AAA CTT GAT CAA CCA GGT GAA AGA TAC CTC GTA     V   V   G   T   N   K   L   D   Q   P   G   E   R   Y   L   V    GAA CAA ACT TTT GTT CAC     E   Q   T   F   V   H    B. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP2 (SEQ ID NOS:54 and 55) is:    TTA GAT GGG CGC ATT GTT GGA GGA CAA GAT GCT GAT ATT GCC AAA TAT     L   D   G   R   I   V   G   G   Q   D   A   D   I   A   K   Y    GGC TAT CAA GCT TCA CTC CAA GTA TTT AAC GAA CAT TTC TGT GGA GCT     G   Y   Q   A   S   L   Q   V   F   N   E   H   F   C   G   A    TCA ATA TTG AAT AAT TAT TGG ATT GTC ACA GCA GCT CAT TGC ATA TAT     S   I   L   N   N   Y   W   I   V   T   A   A   H   C   I   Y    GAT GAA TTC ACG TAT TCA GTT CGA GTC GGC ACC AGT TTC CAA GGA AGA     D   E   F   T   Y   S   V   R   V   G   T   S   F   Q   G   R    CGT GGT TCC GTT CAT CCT GTG GCA CAA ATT ATC AAG CAT CCT GCA TAC     R   G   S   V   H   P   V   A   Q   I   I   K   H   P   A   Y    C. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP4 (SEQ ID NOS:56 and 57) is:    AGG GAA CAA AAG CTG GAG CTC CAC CGC GGT GCG CCG GCT CTA GAA CTA     R   E   Q   K   L   E   L   H   R   G   A   P   A   L   E   L    GTG GAT CCC CCG GGT CTG CAG GAA TTG GCA CGA GGA TGT TCT TGG CTG    V   D   P   P   G   L   Q   E   L   A    R   G   C   S   W   L    TGT TTA GTA GCT ATT CTT TGT GCA GTG GCT GCT GGG CCT ACT AAT CGC     C   L   V   A   I   L   C   A   V   A   A   G   P   T   N   R    ATT GTT GGA GGA TTG GAG GCG AAA AAT GGA ATC ACC CCA TTC ATC GGT     I   V   G   G   L   E   A   K   N   G   I   T   P   F   I   G    TTC TTT GCA AGC GGA AGA CTA TTT CA     F   F   A   S   G   R   L   F    D. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP5 (SEQ ID NOS:58 and 59) is:    ACG AGG TTT CGC TTA GCA ATT GTA TGT GCT CTC GCT GTC TGC ACA TTC    T   R   F   R   L   A   I   V   C   A   L   A   V   C   T   F>    GGT GCC AGT GTT CCA GAA CCA TGG AAA AGA TTA GAT GGT AGA ATC GTA    G   A   S   V   P   E   P   W   K   R   L   D   G   R   I   V>    GGA GGA CAC GAT ACC AGC ATC GAT AAA CAC CCT CAT CAA GTA TCT TTA     G   G   H   D   T   S   I   D   K   H   P   H   Q   V   S   L>    TTG TAC TCC AGC CAC AAT TGT GGT GGT TCC TTG ATT GCC AAA AAC TGG     L   Y   S   S   H   N   C   G   G   S   L   I   A   K   N   W>    GTT TTG ACT GCA GCT CAT TGC ATT GGA GTT AAC AAA TAC AAT GTC CGT     V   L   T   A   A   H   C   I   G   V   N   K   Y   N   V   R>    E. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP6 (SEQ ID NOS:60 and 61) is:    CCC TCA CTA AAG GGA ACA AAA GCT GGA GCT CCA CCG CGG TGC GCC GCT     P   S   L   K   G   T   K   A   G   A   P   P   R   C   A   A    CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG AAT TCG GCA CGA GCG TTT     L   E   L   V   D   P   P   G   C   R   N   S   A   R   A   F    GGT TGG ATT GAG CGC GTC TCA TCT TAC AAG ATA AAG GAT AGA TTA GAT     G   W   I   E   R   V   S   S   Y   K   I   K   D   R   L   D    GGG CGC ATT GTT GGA GGA CAA GAT GCT GAT ATT GCC AAA TAT GGC TAT     G   R   I   V   G   G   Q   D   A   D   I   A   K   Y   G   Y    CAA GCT TCA CTC CAA GTA CTT AAC GAA CAT TTC TGT GGA GCT     Q   A   S   L   Q   V   L   N   E   H   F   C   G   A    F. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP7 (SEQ ID NOS:62 and 63) is:    GCG GTG ATT GTG TCA TTT GTT CTG GCT TGT GCA TTT TCT GTA CAG GCT     A   V   I   V   S   F   V   L   A   C   A   F   S   V   Q   A    CTT CCA TCA AGC AGA ATT GTC AAT GGA CTT GAA GCA GGA GTT GGA CAA     L   P   S   S   R   I   V   N   G   L   E   A   G   V   G   Q    TTT CCA ATT CAG GTT TTC TTA GAC TTG ACA AAT ATC AGA GAC GAA AAA     F   P   I   Q   V   F   L   D   L   T   N   I   R   D   E   K    TCC AGA TGT GGT GGT GCT TTG TTA TCA GAT TCA TGG GTT TTG ACT GCT     S   R   C   G   G   A   L   L   S   D   S   W   V   L   T   A    GCT CAT TGT TTT GAT GAT TTG AAG TCT ATG GTA GTG TCC GTT GGT GCT     A   H   C   F   D   D   L   K   S   M   V   V   S   V   G   A    CAT GAT GTC AGC AAA TCT GAA GAA CCT CAC AGG CAA ACC AGG AAA CCT     H   D   V   S   K   S   E   E   P   H   R   Q   T   R   K   P    GAA     E    G. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP12 (SEQ ID NOS:64 and 65) is:    GTA CTG ATC GTT TTA GCA GTC ATT GAA TTC GCA TCA GCG TCT TCA ATC     V   L   I   V   L   A   V   I   E   F   A   S   A   S   S   I    GGC TGG AGA ATC GTG GGT GGT GAA AAT GCT AAA GAA AAA TCG GTG CCC     G   W   R   I   V   G   G   E   N   A   K   E   K   S   V   P    TAT CAA GTT TCM CTT CGA AAT GCT GAA AAC AAA CAT TTY TGT GGR GGR     Y   Q   V   S   L   R   N   A   E   N   K   H   F   C   G   G    H. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP13 (SEQ ID NOS:66 and 67) is:    TTC GGC TTC AAG CTA AGT CAT TTG GTA AGT AAG TAC TGT GCT TGT GCA     F   G   F   K   L   S   H   L   V   S   K   Y   C   A   C   A    TTA GCA TCG GCA CTG AAG TAC TCC ATC GAT CAT GGT CCT CGT ATC ATC     L   A   S   A   L   K   Y   S   I   D   H   G   P   R   I   I    GGA GGT GAA GTT GCA GGT GAA GGA TCA GCA CCT TAC CAG GTG TCC TTA     G   G   E   V   A   G   E   G   S   A   P   Y   Q   V   S   L    AGA ACC AAG GAA GGA AAT CAT TTT TGC GGT GGA TCA ATA CTA AAT AAG     R   T   K   E   G   N   H   F   C   G   G   S   I   L   N   K    CGA TGG GTT GTA ACT GCA GCA CAT TGT CTT GAA CCG GAA ATA TTA GAT     R   W   V   V   T   A   A   H   C   L   E   P   E   I   L   D    TCG GTA TAC GTC GGA TCC AAT CAC TTA GAC CGA AAA GGC AGA TAT TAC     S   V   Y   V   G   S   N   H   L   D   R   K   G   R   Y   Y    GAC GTA GAA CGG TAT ATA ATT CAT GAA AAA TAT ATA GGA GAA CTA AAT     D   V   E   R   Y   I   I   H   E   K   Y   I   G   E   L   N    AAT TTT TAT GCT GAC ATC GGT CTA ATA AAA CTT GAT GGA AGA CTT AGA     N   F   Y   A   D   I   G   L   I   K   L   D   G   R   L   R    ATT CAA     I   Q    I. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP14 (SEQ ID NOS:68 and 69) is:    CGG GCT GCA GGA ATT CGG CAC GAG AAG AAA CTG CCA ATA TTA ATC GCC     R   A   A   G   I   R   H   E   K   K   L   P   I   L   I   A    TTG ATC GGA TGC GTT CTT TCT GAA GAA ATA GAG GAT CGC ATT GTC GGC     L   I   G   C   V   L   S   E   E   I   E   D   R   I   V   G    GGA ACG GCA GTT GAT ATA AGA GGT TTT CCC TGG CAG GTA TCA ATT CAA     G   T   A   V   D   I   R   G   F   P   W   Q   V   S   I   Q    ACC GAA AAC CGT CAT TTT TGT GGT GGT TCT ATT ATC GAT AAA AGC TGG     T   E   N   R   H   F   C   G   G   S   I   I   D   K   S   W    ATA TTA ACT GCC GCA CAT TGT GTA CMC GAT ATG AAG ATG TCG AAC TGG     I   L   T   A   A   H   C   V   X   D   M   K   M   S   N   W    J. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP15 (SEQ ID NOS:70 and 71) is:    CAC GAG ATT TTA TTA AGC GCA TTA TTT GCA AGT GTA ATT TGC TCC TTT     H   E   I   L   L   S   A   L   F   A   S   V   I   C   S   F    AAC GCG GAA GTA CAA AAT CGA ATC GTT GGT GGC AAT GAT GTA AGT ATT     N   A   E   V   Q   N   R   I   V   G   G   N   D   V   S   I    TCA AAA ATT GGG TGG CAA GTA TCT ATT VAA AGT AAT AAA CAA CAT TTC     S   K   I   G   W   Q   V   S   I   Q   S   N   K   Q   H   F    TGT GGT GGT TCA ATC ATT GCT AAA GAT GGG TCC     C   G   G   S   I   I   A   K   D   G   S    K. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP16 (SEQ ID NOS:72 and 73) is:    ATC ATG GCA AAT TTT AGG CTA TTC ACC TTA CTA GCC TTG GTT GCA GTA     I   M   A   N   F   R   L   F   T   L   L   A   L   V   S   V    GCA ACT TCC AAA TAT ATT GAT CCA AGA ATA ATT GGA GGC GAA GAT GCT     A   T   S   K   Y   I   D   P   R   I   I   G   G   E   D   A    CCT GAA GGC TCG GCT CCG TAC CAA GTT TCA TTG AGA AAT CAG AAT CTG     P   E   G   S   A   P   Y   Q   V   S   L   R   N   Q   N   L    GAG CAT TTC TGT GGT GGT TCC ATT     E   H   F   C   G   G   S   I    L. The apparent N-terminal nucleic acid and deduced amino acid    sequence of nfSP17 (SEQ ID NOS:74 and 75) is:    GCA CGA GAT CGC ATT GTT GGA GGA TTG GAG GCG AAA AAT GGA TCA GCC     A   R   D   R   I   V   G   G   L   E   A   K   N   G   S   A    CCA TTC ATG GTT TCT TTG CAA GCG GAA GAC TAT TTT CAT TTT TGT GGA     P   F   M   V   S   L   Q   A   E   D   Y   F   H   F   C   G    TCC TCT ATT CTG AAT GAG AGA TGG GTT CTT ACT GCT GCT CAC TGT ATC     S   S   I   L   N   E   R   W   V   L   T   A   A   H   C   I    CAA CCA AAT GTA CAC AAG TAC GTT TAC GTC GGT TCG AAC AAC GTA GAA     Q   P   N   V   H   K   Y   V   Y   V   G   S   N   N   V   E    M. The apparent C-terminal nucleic acid and deduced amino acid    sequence of nfSP12 (SEQ ID NOS:76 and 77) is:    CCA ATC CAC GAT AGC CAA TAT GCA CTT TTG CAG ATA TGG GTC AAG GGT     P   I   H   D   S   Q   Y   A   L   L   Q   I   W   V   K   G>    GCA TGT AAG GGT GAT TCC GGT GGC CCC TTA GTC ATC AAT GGA CAA CTT     A   C   K   G   D   S   G   G   P   L   V   I   N   G   Q   L>    CAT GGA ATT GTT TCC TGG GGC ATT CCT TGC GCT GTC GCA AGC CTG ATG     H   G   I   V   S   W   G   I   P   C   A   V   A   S   L   M>    TAT TCA CAA GAG TTT CTC ATT ATG TCG ATT GGA TTA AAT CCA AAA TTG     Y   S   Q   E   F   L   I   M   S   I   G   L   N   P   K   L>    AAT AAA ATT GTT TAG     N   K   I   V   *    N. The apparent C-terminal nucleic acid and deduced amino acid    sequence of nfSP13 (SEQ ID NOS:78 and 79) is (the initial GGPL    is next to the conserved active-site serine):    GGA GGT CCT TTG GCA ATC AAT GGT GAA CTT GTT GGT GTT ACT TCA TTC     G   G   P   L   A   I   N   G   E   L   V   G   V   T   S   F    ATT ATG GGG ACA TGT GGA GGA GGA CAT CCT GAT GTC TTC GGT CGA GTC     I   M   G   T   C   G   G   G   H   P   D   V   F   G   R   V    CTT GAC TTC AAA CCA TGG ATT GAT TCT CAT ATG GCA AAT GAC GGC GCT     L   D   F   K   P   W   I   D   S   H   M   A   N   D   G   A    AAT TCT TTT ATT TAA     N   S   F   I   *    __________________________________________________________________________

Example 12

This Example describes the purification of a flea aminopeptidase of thepresent invention.

The starting material for the isolation of a flea aminopeptidase was aflea midgut lysate preparation that had been depleted of serineproteases by passage over a benzamidine-Sepharose affinity column. Toassay for aminopeptidase activity, the synthetic substrate L-Leucine-AMC(Leu-AMC), which releases a fluorescent AMC leaving group uponproteolytic cleavage, was incubated with the serine protease-depletedflea midgut preparation. Aminopeptidase activity was easily detectablewith as little as 1.2 μg of lysate, both confirming the presence of anaminopeptidase (as indicated in other Examples herein) and allowing forthe detection of aminopeptidase activity in fractions collectedthroughout subsequent fractionation and purification procedures.

Serine protease-depleted flea midgut lysates (samples of about 1.2 μgand about 12 μg) were incubated with Leu-AMC in the presence of thefollowing inhibitors: 1 mM pefabloc, 1 mg/ml trypsin/chymotrypsininhibitor, 1 mg/ml trypsin inhibitor, 1 mM TPCK, 1 μg/ml pepstatin, 10μg/ml E-64, 10 μg/ml of leupeptin, 10 mM EDTA, and 86 μg/ml of bestatin.Only bestatin inhibited the flea protease that cleaved Leu-AMC, whereasboth EDTA and bestatin inhibited the control protease, a leucineaminopeptidase. These results indicated that the flea protease beingcharacterized was an aminopeptidase, but apparently was not ametallo-aminopeptidase like the "classic" leucine aminopeptidases.

A flea aminopeptidase was purified using the following protocol. Fleamidgut lysates cleared of serine protease activity were fractionated byanion-exchange chromatography. Those fractions containing aminopeptidaseactivity were pooled and subjected to cation-exchange chromatography,and the resulting fractions were again assayed for activity withL-Leu-AMC in 96-well plates.

Fractions containing aminopeptidase activity were subjected to SDS-PAGEand silver-stained to identify the protein(s) exhibiting that activity.Aminopeptidase activity was found to be associated with proteins thatmigrated at a molecular weight of about 95 kD and about 56 kD whensubjected to SDS-PAGE. The 95 kD and 56 kD proteins may each beaminopeptidases or they may be subunits of a larger enzyme. A number ofknown aminopeptidases are multi-subunit enzymes comprised of subunitsranging from about 45 kD to about 55 kD and from about 90 kD to about 95kD.

Additional purification studies have indicated that the majority ofaminopeptidase activity was found to be associated with the membranepellet preparation and could be solubilized with detergent.Aminopeptidase activity in such preparations was also monitored duringpurification using L-Leu-AMC, and appeared to be associated with the 95kD and 56 kD proteins when active fractions were analyzed by SDS-PAGEand silver staining. The 95 kD and 56 kD protein were co-purified togreater than 90% purity by cation exchange chromatography, affinitychromatography using w-aminohexyl agarose, and C-4 reverse phasechromatography. N-terminal amino acid sequence analysis indicated thatboth isolated aminopeptidases appeared to be blocked at the aminoterminus.

Example 13

This Example describes the isolation of a flea aminopeptidase nucleicacid molecule of the present invention

A nucleic acid molecule encoding a flea aminopeptidase was isolated inthe following manner. A DNA fragment was PCR amplified from a whole fedflea cDNA expression library (prepared as described in Example 8 usingdegenerate primers, the design of which was based on conserved regionsof bovine lens leucine aminopeptidase (LAP). The specific LAP-basedprimers used included: degenerate LAP sense primer A, corresponding tobovine lens LAP amino acid sequence from about amino acid 247 through257 and having nucleic acid sequence 5' GTW GGW AAA GGW WTW ACW TTY GATTCW GGW GG 3', represented herein as SEQ ID NO:48; and degenerate LAPantisense primer C, corresponding to bovine lens LAP amino acid sequencefrom about amino acid 335 through 329 and having nucleic acid sequence5' CG WCC TTC WGC ATC WGT ATT 3', represented herein as SEQ ID NO:49.Also used were vector primers having SEQ ID NO:13 and SEQ ID NO:14,described in Example 11.

In a first experiment, the LAP primer C having SEQ ID NO:49 and the M13reverse vector primer having SEQ ID NO:13 were used to PCR amplify DNAfragments from the expression library. The resultant PCR products werescreened by hybridization under standard hybridization conditions withLAP primer A having SEQ ID NO:48. A PCR product that hybridized with SEQID NO:48 was subjected to nested (actually semi-nested) PCRamplification using LAP primer C and the T3 vector primer having SEQ IDNO:14. The resulting PCR product, which was about 900 nucleotides inlength (denoted nfAP₉₀₀) and hybridized under standard (i.e., stringent)hybridization conditions with LAP primer A, was cloned into the TA™vector and analyzed by DNA sequence analysis as described in Example 11.

The nucleic acid sequence of a portion of nfAP₉₀₀, namely of nfAP₄₅₃, isrepresented herein as SEQ ID NO:50. Translation of SEQ ID NO:50 yields aprotein of about 151 amino acids, denoted herein as PfAP₁₅₁, the aminoacid sequence of which is represented herein as SEQ ID NO:51. Analysisof SEQ ID NO:51 suggests that the sequence includes a leader segment ofabout 15 amino acids followed by a mature protein that has about 32%identity with the bovine lens LAP. The corresponding bovine and fleanucleotide sequences are about 29% identical.

Example 14

This Example describes the production of an anti-flea midgut proteaseantiserum and its use to inhibit flea protease activity therebysupporting the utility of protease-based vaccines as anti-flea agents.

Anti-flea protease antiserum was produced in the following manner. Arabbit was immunized 3 times with approximately 40-50 μg of a fleamidgut protease preparation that had been affinity-purified usingbenzamidine sepharose as described in Example 7 and then combined withFreund's complete adjuvant for the first immunization and withincomplete adjuvant for the second and third immunizations according tostandard procedures. After the second immunization, endpoint titers ofaround 1:3200 were obtained, while the third immunization boosted theanti-protease titers to about 1:6400. Western blot analysis of theimmunoreactivity of the resultant anti-flea protease antiserum againstthe affinity-purified midgut protease preparation demonstrated thepresence of at least 7-8 reactive protease bands. This was an importantobservation since there are numerous reports in the literature ofdifficulties associated with generating high-titered antisera againstcertain classes of proteases.

To assess the inhibitory activity of the rabbit anti-flea proteaseantiserum against flea midgut proteases, an in vitro assay whichmeasures trypsin activity as a function of absorbance at OD₄₅₀ using adefined protein substrate (i.e., succinylated casein) was establishedusing a commercially available kit (available from Pierce, Rockford,Ill.). In preliminary assays, the proteolytic activity of theaffinity-purified flea midgut protease preparation was about 25-30% ofthe activity observed with the trypsin control. This lower activity isnot unexpected since the flea proteases may require different reactionconditions than the trypsin control for optimal activity. Also, theprimary amino acid sequences determined for the flea proteases asdescribed in Examples 7, 10 and 11 are suggestive of highly specializedfunctions that may require specific substrates for determining optimalactivity. Incubation of the affinity purified midgut proteasepreparation with the succinylated casein substrate in the presence ofabout 500 ng of the rabbit anti-flea protease antibody-containing serumcollected after the second immunization reduced the proteolytic activityof the protease preparation by about 20%. This result, using asuboptimal assay, supports the feasibility of using immunologicalmethods to inhibit flea midgut protease activity.

Using a similar immunization protocol, anti-flea protease antiserum hasalso been generated in cats that has exhibited immunoreactivity, asidentified by Western blot analysis, against several proteases in theaffinity-purified midgut protease preparation. The cat anti-fleaprotease antiserum also reduced proteolytic activity of an affinitypurified midgut protease preparation by about 20%, using the same assayas described for analyzing the rabbit antiserum.

Example 15

This Example demonstrates that flea larvae have predominantlyserine-type proteases.

Newly hatched flea larvae were raised in colony rearing dishes and fedon larval rearing media containing dried bovine blood using standardtechniques. About 300 to 500 larvae were collected at differentdevelopmental stages, homogenized by sonication in flea gut dissectionbuffer (50 mM Tris, 100 mM CaCl₂, pH 8.0) and centrifuged to pellet celldebris. About 25 larval equivalents were incubated with about 2.5 μCi1,3-³ H!-diisopropylfluorophosphate (DFP) overnight at 4° C. Afterincubation, about 10 larval equivalents were spotted onto filter paper,precipitated with 10% trichloroacetic acid, and counted in a liquidscintillation counter. Reducing SDS-PAGE was performed on samplescomprising about 2.5 larval equivalents, and autoradiography wasperformed using standard techniques. In addition, adult flea midgutproteases were extracted and ³ H-labeled in the same manner and examinedby SDS-PAGE and autoradiography. Analysis of the gel indicated that,based on their ability to be labeled with DFP, larval proteases appearto be predominantly serine-type proteases, the production of whichappears to be induced by blood feeding as occurs in adults. Blood-fed3rd instars appeared to have the highest amount of proteolytic activity.

Example 16

The Example demonstrates that flea feces has proteolytic activity, thatis predominantly due to serine proteases.

Flea feces were collected from fleas fed in flea cages placed on a livecat or in a flea feeding system as described in Example 6 in which thefleas were fed bovine blood. Fresh feces were collected every 9-17hours, resuspended in water at 150 mg feces/ml, and centrifuged topellet insoluble material. The soluble fractions were then assayed usingtwo techniques. Western blot analysis was performed on samples subjectedto reducing SDS-PAGE, each lane having about 40 μg of protein. Blottedproteins were incubated overnight at 4° C. with 1:500 rabbit anti-fleaprotease antiserum produced as described in Example 14. Goat anti-rabbitsecondary antibody was used at 1:2000 to develop the Western blot.Analysis of the Western blot indicated the presence of serine-typeproteases in flea feces. Appearance of such proteases migrating at about25 to about 30 kD in such a system suggests the presence of full-lengthserine proteases.

Zymogram analysis was performed by loading approximately 50 μg proteininto each lane of the electrophoresis gel in non-reducing sample buffer.After electrophoresis, the zymogram gel was soaked in 2.5% Triton-X-100to renature the samples and developed at 37° C. in 50 mM Tris, pH 7.6,200 mM NaCl, 5 mM CaCl₂, 0.02% Brij 35. Coomassie staining the gelrevealed clear plaques where active proteases digested the gelatin inthe gel matrix. Both of these techniques indicated the presence ofserine-type proteases in flea feces.

Example 17

This Example demonstrates that fleas that have fed onantibody-containing blood have antibodies in their feces, suggesting animmunological method to eradicate flea larvae, which feed from fleafeces.

The ability of antibodies in a blood meal to be taken up by fleas, passthrough the midgut and be excreted in the feces was demonstrated in thefollowing manner. A commercially available rabbit antibody againstovalbumin was added at near physiological concentration (i.e., at about2 mg/ml) to the blood meal of adult fleas in a flea feeding system asdescribed in Example 6. Feces were collected at 24 hr and 48 hr afterfeeding and rehydrated in phosphate saline buffer. The rehydrated fecalsamples were subjected to Western blot analysis and shown to containrabbit anti-ovalbumin antibodies that were apparently full-length, usinga rabbit-specific secondary antibody screen against the Fc region ofrabbit antibodies. Supernatants of flea midguts collected at the sametime periods showed residual amounts of rabbit anti-ovalbuminantibodies.

In a second experiment, fleas were fed in a similar manner a blood mealcontaining cat-specific antiserum generated against keyhole limpethemocyanin (KLH) and feces were collected at 24, 48 and 72 hourspost-feeding. The sample collected at 24 hours was divided into halves,with one half rehydrated immediately in PBS while the second half wasrehydrated 7 days later. Fecal samples collected at 48 and 72 hours wereheld for 6 and 5 days, respectively, after collection in desiccated formprior to rehydration. Aliquots of the bloodmeal containing the KLHantiserum fed to the fleas were also sampled at 1 and 2 days. All of therecovered antibodies were reactive against KLH by Western blot analysis,with a pattern or reactivity indistinguishable from the cat anti-KLHserum alone.

These studies demonstrate that antibodies are able to pass through theflea midgut in intact form and are able to maintain theirantigen-binding characteristics, thereby supporting the feasibility ofan immunological method to target larval development, since flea larvaein their normal habitat feed from flea feces.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 79    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 32 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    - Ile Ile Gly Gly Glu Val Ala Gly Glu Gly Se - #r Ala Pro Tyr Gln Val    #                15    - Ser Leu Arg Thr Lys Glu Gly Asn His Phe Se - #r Gly Gly Ser Ile Leu    #            30    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 17 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    - Xaa Val Gly Gly His Asp Thr Ser Ile Asp Xa - #a His Pro His Gln Val    #                15    - Thr    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 22 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Ala Asp Ala Ala Pro Gly As - #n Ala Pro Phe Gln Val    #                15    - Ser Leu Arg Asp Lys Gly                20    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 34 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Gln Asp Ala Asp Ile Ala Ly - #s Tyr Gly Tyr Gln Ala    #                15    - Ser Leu Gln Val Phe Asn Glu His Phe Xaa Gl - #y Ala Xaa Ile Leu Asn    #            30    - Asn Tyr    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 25 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Thr Asp Val Asn Ile Glu As - #n Phe Gly Trp Gln Val    #                15    - Ser Leu Phe Asp Arg Asn Gly His Phe    #            25    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 22 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly His Asp Thr Ser Ile Asp Ly - #s His Pro Phe Gln Val    #                15    - Ser Leu Ile Asp Lys Asn                20    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 23 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ    - Val Val Gly Gly Leu Glu Ala Ala Glu Gly Se - #r Ala Pro Tyr Gln Val    #                15    - Xaa Leu Gln Trp Gly Asn Phe                20    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Glu Asp Ala Glu Leu Gly Gl - #u Xaa Pro Thr Gln    #                15    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ    - Asp Glu Asp Gly Lys Asp Asp Ser Ala Pro Gl - #y Glu Ile    #                10    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ    - Cys Xaa Gly Asp Ser Gly Gly Pro Leu    1               5    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (primer)    #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ    #             27   TCTC CCTGGCA    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (primer)    #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ    #                24CCTT GACA    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (primer)    #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ    # 19               ATG    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (primer)    #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ    #   17             G    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (probe)    #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ    #                23CWCA TTG    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 672 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..672    #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ    - GCA CGA GAT CGC ATT GTT GGA GGA TTG GAG GC - #G AAA AAT GGA TCA GCC      48    Ala Arg Asp Arg Ile Val Gly Gly Leu Glu Al - #a Lys Asn Gly Ser Ala    #                 15    - CCA TTC ATG GTT TCT TTG CAA GCG GAA GAC TA - #T TTT CAT TTT TGT GGA      96    Pro Phe Met Val Ser Leu Gln Ala Glu Asp Ty - #r Phe His Phe Cys Gly    #             30    - TCC TCT ATT CTG AAT GAG AGA TGG GTT CTT AC - #T GCT GCT CAC TGT ATC     144    Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Th - #r Ala Ala His Cys Ile    #         45    - CAA CCA AAT GTA CAC AAG TAC GTT TAC GTC GG - #T TCG AAC AAC GTA GAA     192    Gln Pro Asn Val His Lys Tyr Val Tyr Val Gl - #y Ser Asn Asn Val Glu    #     60    - GTA GGC GGA ACA CAC TAC GAA ATC GAA AAA GC - #T TTC TAT CAC GAA GAA     240    Val Gly Gly Thr His Tyr Glu Ile Glu Lys Al - #a Phe Tyr His Glu Glu    # 80    - TAT GAT GGA GTA GAT CTT GTA GAT CAT GAT GT - #G ATT GAT CAA AGT GAG     288    Tyr Asp Gly Val Asp Leu Val Asp His Asp Va - #l Ile Asp Gln Ser Glu    #                 95    - ACA AAC ATT GAT TTA ATG AAG TGT CAA CCC AT - #T AAA TTA CGA AGA AAG     336    Thr Asn Ile Asp Leu Met Lys Cys Gln Pro Il - #e Lys Leu Arg Arg Lys    #           110    - CCA CTC GTT GGT GGT GAG GAA TTG AGA GCA GT - #A GGC TGG GGA AAT ACA     384    Pro Leu Val Gly Gly Glu Glu Leu Arg Ala Va - #l Gly Trp Gly Asn Thr    #       125    - AAT TCA GCA GGG GAA AAT TTT CCA TTG AAA CT - #T CAA GAA TTG TAC GTG     432    Asn Ser Ala Gly Glu Asn Phe Pro Leu Lys Le - #u Gln Glu Leu Tyr Val    #   140    - AAA GCT TTG ACT AAT GAG GAG TGC AAA GCT AA - #A TCA CCA ATT CCA CCA     480    Lys Ala Leu Thr Asn Glu Glu Cys Lys Ala Ly - #s Ser Pro Ile Pro Pro    145                 1 - #50                 1 - #55                 1 -    #60    - ACG ACC CAA GTC TGC ACA CTT TTG GAA AAG AA - #T CAT GGT GTA TGC TCG     528    Thr Thr Gln Val Cys Thr Leu Leu Glu Lys As - #n His Gly Val Cys Ser    #               175    - GGA GAT TCT GGT GGT CCA TTG CTT TTG GAT GG - #C GAG CAA GTT GGC ATT     576    Gly Asp Ser Gly Gly Pro Leu Leu Leu Asp Gl - #y Glu Gln Val Gly Ile    #           190    - GCC TCA TTT GTT ATC TTC AAA TGC GCA ATG GG - #G TAC CCT GAC TAT TTC     624    Ala Ser Phe Val Ile Phe Lys Cys Ala Met Gl - #y Tyr Pro Asp Tyr Phe    #       205    - ACA AGA TTG TCT CTA TAT GTA GAT TGG ATT GA - #A CAA CAC ATG GAT TAA     672    Thr Arg Leu Ser Leu Tyr Val Asp Trp Ile Gl - #u Gln His Met Asp  *    #   220    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  223 ami - #no acids              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:17:    - Ala Arg Asp Arg Ile Val Gly Gly Leu Glu Al - #a Lys Asn Gly Ser Ala    #                 15    - Pro Phe Met Val Ser Leu Gln Ala Glu Asp Ty - #r Phe His Phe Cys Gly    #             30    - Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Th - #r Ala Ala His Cys Ile    #         45    - Gln Pro Asn Val His Lys Tyr Val Tyr Val Gl - #y Ser Asn Asn Val Glu    #     60    - Val Gly Gly Thr His Tyr Glu Ile Glu Lys Al - #a Phe Tyr His Glu Glu    # 80    - Tyr Asp Gly Val Asp Leu Val Asp His Asp Va - #l Ile Asp Gln Ser Glu    #                 95    - Thr Asn Ile Asp Leu Met Lys Cys Gln Pro Il - #e Lys Leu Arg Arg Lys    #           110    - Pro Leu Val Gly Gly Glu Glu Leu Arg Ala Va - #l Gly Trp Gly Asn Thr    #       125    - Asn Ser Ala Gly Glu Asn Phe Pro Leu Lys Le - #u Gln Glu Leu Tyr Val    #   140    - Lys Ala Leu Thr Asn Glu Glu Cys Lys Ala Ly - #s Ser Pro Ile Pro Pro    145                 1 - #50                 1 - #55                 1 -    #60    - Thr Thr Gln Val Cys Thr Leu Leu Glu Lys As - #n His Gly Val Cys Ser    #               175    - Gly Asp Ser Gly Gly Pro Leu Leu Leu Asp Gl - #y Glu Gln Val Gly Ile    #           190    - Ala Ser Phe Val Ile Phe Lys Cys Ala Met Gl - #y Tyr Pro Asp Tyr Phe    #       205    - Thr Arg Leu Ser Leu Tyr Val Asp Trp Ile Gl - #u Gln His Met Asp    #   220    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 156 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..156    #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ    - GGG TGG GGA AGA CTT GGA GCT AAC TTG AAT GG - #A CCG AAT GAA CTC CAA      48    Gly Trp Gly Arg Leu Gly Ala Asn Leu Asn Gl - #y Pro Asn Glu Leu Gln    #                 15    - GAA CTT AAC ACT GTC ACA TTA AGC CAC CAG CA - #A TGT GTA AGA CAA CAA      96    Glu Leu Asn Thr Val Thr Leu Ser His Gln Gl - #n Cys Val Arg Gln Gln    #             30    - ATT TAT CCA GTA TAC GAC AGC CAA CTT TGC AC - #A TTT GTT GGC AGT GGA     144    Ile Tyr Pro Val Tyr Asp Ser Gln Leu Cys Th - #r Phe Val Gly Ser Gly    #         45    #      156    Arg Gly Ala Cys         50    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 52 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:19:    - Gly Trp Gly Arg Leu Gly Ala Asn Leu Asn Gl - #y Pro Asn Glu Leu Gln    #                 15    - Glu Leu Asn Thr Val Thr Leu Ser His Gln Gl - #n Cys Val Arg Gln Gln    #             30    - Ile Tyr Pro Val Tyr Asp Ser Gln Leu Cys Th - #r Phe Val Gly Ser Gly    #         45    - Arg Gly Ala Cys         50    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 168 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..168    #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA TGG GGC AAA TTA AGT GAA TCA GGA CCC AA - #G CCA GTA AAT CTA CAA      48    Gly Trp Gly Lys Leu Ser Glu Ser Gly Pro Ly - #s Pro Val Asn Leu Gln    #                 15    - GGA GTA AAA GTG CCT TAT GTG ACC AAG ATA CA - #T GCT CTG ACA GCT ACG      96    Gly Val Lys Val Pro Tyr Val Thr Lys Ile Hi - #s Ala Leu Thr Ala Thr    #             30    - TCT TTG CAG GTA AAA GAT ATC ACC GAA AAC AT - #G TTG TGT GCC GGA GTT     144    Ser Leu Gln Val Lys Asp Ile Thr Glu Asn Me - #t Leu Cys Ala Gly Val    #         45    #               168AG GAC TCC TGC    Arg Arg Gly Gly Lys Asp Ser Cys    #     55    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 56 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:21:    - Gly Trp Gly Lys Leu Ser Glu Ser Gly Pro Ly - #s Pro Val Asn Leu Gln    #                 15    - Gly Val Lys Val Pro Tyr Val Thr Lys Ile Hi - #s Ala Leu Thr Ala Thr    #             30    - Ser Leu Gln Val Lys Asp Ile Thr Glu Asn Me - #t Leu Cys Ala Gly Val    #         45    - Arg Arg Gly Gly Lys Asp Ser Cys    #     55    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 177 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..177    #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA TGG GGA TCA AGA TCT ACT TCC AAT TTC CC - #A TCT TAC CCC AAC CTT      48    Gly Trp Gly Ser Arg Ser Thr Ser Asn Phe Pr - #o Ser Tyr Pro Asn Leu    #                 15    - TTA CAG ACC GTT GAC AAA CCA ATT GTA TCT TA - #T GCC GAA TGT GAG AAA      96    Leu Gln Thr Val Asp Lys Pro Ile Val Ser Ty - #r Ala Glu Cys Glu Lys    #             30    - GTA TTG GGA GGT CCT GGA GCC TCA CCA CTT CA - #C CCC TTG AAC CTC TGC     144    Val Leu Gly Gly Pro Gly Ala Ser Pro Leu Hi - #s Pro Leu Asn Leu Cys    #         45    #        177C TTG ACC GGT GGA GTA AGC GCT TG - #T    Thr Gly Pro Leu Thr Gly Gly Val Ser Ala Cy - #s    #     55    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 59 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:23:    - Gly Trp Gly Ser Arg Ser Thr Ser Asn Phe Pr - #o Ser Tyr Pro Asn Leu    #                 15    - Leu Gln Thr Val Asp Lys Pro Ile Val Ser Ty - #r Ala Glu Cys Glu Lys    #             30    - Val Leu Gly Gly Pro Gly Ala Ser Pro Leu Hi - #s Pro Leu Asn Leu Cys    #         45    - Thr Gly Pro Leu Thr Gly Gly Val Ser Ala Cy - #s    #     55    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 156 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..156    #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ    - GGC TGG GGA AAT ACA AAT TCA GCA GGG GAA AA - #T TTT CCA TTG AAA CTT      48    Gly Trp Gly Asn Thr Asn Ser Ala Gly Glu As - #n Phe Pro Leu Lys Leu    #                 15    - CAA GAA TTG TAC GTG AAA GCT TTG ACT AAT GA - #G GAG TGC AAA GCT AAA      96    Gln Glu Leu Tyr Val Lys Ala Leu Thr Asn Gl - #u Glu Cys Lys Ala Lys    #             30    - TCA CCA ATT CCA CCA ACG ACC CAA GTC TGC AC - #A CTT TTG GAA AAG AAT     144    Ser Pro Ile Pro Pro Thr Thr Gln Val Cys Th - #r Leu Leu Glu Lys Asn    #         45    #      156    His Gly Val Cys         50    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 52 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:25:    - Gly Trp Gly Asn Thr Asn Ser Ala Gly Glu As - #n Phe Pro Leu Lys Leu    #                 15    - Gln Glu Leu Tyr Val Lys Ala Leu Thr Asn Gl - #u Glu Cys Lys Ala Lys    #             30    - Ser Pro Ile Pro Pro Thr Thr Gln Val Cys Th - #r Leu Leu Glu Lys Asn    #         45    - His Gly Val Cys         50    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 159 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..159    #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA TGG GGA TCA ACT GGA TCT GGT GGT CCA AT - #T ACA AAT GTT CTA CAA      48    Gly Trp Gly Ser Thr Gly Ser Gly Gly Pro Il - #e Thr Asn Val Leu Gln    #                 15    - GAA GTC GAA GTT CCA TTT ATC GAC TTC AAC AC - #C TGC CGA AAA TCC TAC      96    Glu Val Glu Val Pro Phe Ile Asp Phe Asn Th - #r Cys Arg Lys Ser Tyr    #             30    - TCA ACC AGC TTA ACC GAC CGT ATG TTC TGC GC - #T GGA TTT TTG GGA ATT     144    Ser Thr Ser Leu Thr Asp Arg Met Phe Cys Al - #a Gly Phe Leu Gly Ile    #         45    #   159            GC    Gly Gly Lys Ala Cys         50    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 53 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:27:    - Gly Trp Gly Ser Thr Gly Ser Gly Gly Pro Il - #e Thr Asn Val Leu Gln    #                 15    - Glu Val Glu Val Pro Phe Ile Asp Phe Asn Th - #r Cys Arg Lys Ser Tyr    #             30    - Ser Thr Ser Leu Thr Asp Arg Met Phe Cys Al - #a Gly Phe Leu Gly Ile    #         45    - Gly Gly Lys Ala Cys         50    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 168 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..168    #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ    - GGC TGG GGA AAT TTA GGG GAA GAT GAG GAC GA - #C CCC GAA CAA CTG CAA      48    Gly Trp Gly Asn Leu Gly Glu Asp Glu Asp As - #p Pro Glu Gln Leu Gln    #                 15    - TAT GTA AAG GTA CCT ATT GTT AAC TGG ACT CA - #G TGC AAA ACT ATA TAT      96    Tyr Val Lys Val Pro Ile Val Asn Trp Thr Gl - #n Cys Lys Thr Ile Tyr    #             30    - GGA AAT GAA GGA CTA ATA ATT ACC CAA AAT AT - #G ATT TGT GCT GGT TAT     144    Gly Asn Glu Gly Leu Ile Ile Thr Gln Asn Me - #t Ile Cys Ala Gly Tyr    #         45    #               168AG GAC TCT TGC    Pro Asp Gly Gly Lys Asp Ser Cys    #     55    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 56 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:29:    - Gly Trp Gly Asn Leu Gly Glu Asp Glu Asp As - #p Pro Glu Gln Leu Gln    #                 15    - Tyr Val Lys Val Pro Ile Val Asn Trp Thr Gl - #n Cys Lys Thr Ile Tyr    #             30    - Gly Asn Glu Gly Leu Ile Ile Thr Gln Asn Me - #t Ile Cys Ala Gly Tyr    #         45    - Pro Asp Gly Gly Lys Asp Ser Cys    #     55    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 159 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..159    #ID NO:30:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA TGG GCA TCT CCA AAG ATT TCC CCT GCT TT - #C GAA TTG CCT GAC AAA      48    Gly Trp Ala Ser Pro Lys Ile Ser Pro Ala Ph - #e Glu Leu Pro Asp Lys    #                 15    - CTA CAG TAC ACA ACT TTG GAA GTC CAA CCA AG - #T GAA GAC TGC AAA AAA      96    Leu Gln Tyr Thr Thr Leu Glu Val Gln Pro Se - #r Glu Asp Cys Lys Lys    #             30    - GTA TGG GCC CCT TAC ATG CGC GAC TAC ATC CT - #T TGT GCC AAA TTT GAA     144    Val Trp Ala Pro Tyr Met Arg Asp Tyr Ile Le - #u Cys Ala Lys Phe Glu    #         45    #   159            GC    Lys Gln Asn Ile Cys         50    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 53 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:31:    - Gly Trp Ala Ser Pro Lys Ile Ser Pro Ala Ph - #e Glu Leu Pro Asp Lys    #                 15    - Leu Gln Tyr Thr Thr Leu Glu Val Gln Pro Se - #r Glu Asp Cys Lys Lys    #             30    - Val Trp Ala Pro Tyr Met Arg Asp Tyr Ile Le - #u Cys Ala Lys Phe Glu    #         45    - Lys Gln Asn Ile Cys         50    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 186 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..186    -     (ix) FEATURE:    #Val, Ala, Asp, Glu or Glya =              (B) LOCATION: 29    #ID NO:32:(xi) SEQUENCE DESCRIPTION: SEQ    - GGT TGG GGA AAG ATA GAC TAT TCT GAG AGC AG - #A AGT GAT GAC CTA CTG      48    Gly Trp Gly Lys Ile Asp Tyr Ser Glu Ser Ar - #g Ser Asp Asp Leu Leu    #                 15    - AAA GTA GTA CTG AAA ATT ATT GAT AAT AGG CA - #A TGC GVY CCC TTA TAC      96    Lys Val Val Leu Lys Ile Ile Asp Asn Arg Gl - #n Cys Xaa Pro Leu Tyr    #             30    - GTT GAT CAG ATT AAT AGA AGA AGA TTG AGA AA - #T GGA ATT GTA GAA ACA     144    Val Asp Gln Ile Asn Arg Arg Arg Leu Arg As - #n Gly Ile Val Glu Thr    #         45    - CAG ATG TGT GCA GGA GAA TTG GAT GGT GGA AA - #A GAC ACT TGC    # 186    Gln Met Cys Ala Gly Glu Leu Asp Gly Gly Ly - #s Asp Thr Cys    #     60    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 62 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:    #Val, Ala, Asp, Glu or Glya =              (B) LOCATION: 29    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:33:    - Gly Trp Gly Lys Ile Asp Tyr Ser Glu Ser Ar - #g Ser Asp Asp Leu Leu    #                 15    - Lys Val Val Leu Lys Ile Ile Asp Asn Arg Gl - #n Cys Xaa Pro Leu Tyr    #             30    - Val Asp Gln Ile Asn Arg Arg Arg Leu Arg As - #n Gly Ile Val Glu Thr    #         45    - Gln Met Cys Ala Gly Glu Leu Asp Gly Gly Ly - #s Asp Thr Cys    #     60    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 168 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..168    #ID NO:34:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA TGG GGA AGA ACA TCG TTC GGT GGC CAA TT - #G TCT AAA AAT CTG CGA      48    Gly Trp Gly Arg Thr Ser Phe Gly Gly Gln Le - #u Ser Lys Asn Leu Arg    #                 15    - GGA GTC GAG TTG GAA ATA ATA GAT CTA TTC GA - #T TGT TTC CTT TCC TAC      96    Gly Val Glu Leu Glu Ile Ile Asp Leu Phe As - #p Cys Phe Leu Ser Tyr    #             30    - ATG GAT AAA GTA AAC GTG TCC GAA AGG CAA GT - #T TGC GCT GGA ATC CCC     144    Met Asp Lys Val Asn Val Ser Glu Arg Gln Va - #l Cys Ala Gly Ile Pro    #         45    #               168AA GAT TCT TGC    Val Val Gly Gly Lys Asp Ser Cys    #     55    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 56 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:35:    - Gly Trp Gly Arg Thr Ser Phe Gly Gly Gln Le - #u Ser Lys Asn Leu Arg    #                 15    - Gly Val Glu Leu Glu Ile Ile Asp Leu Phe As - #p Cys Phe Leu Ser Tyr    #             30    - Met Asp Lys Val Asn Val Ser Glu Arg Gln Va - #l Cys Ala Gly Ile Pro    #         45    - Val Val Gly Gly Lys Asp Ser Cys    #     55    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 120 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..120    #ID NO:36:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA TGG GGT GCA GTC TAC GAA GGA GGT GCA GG - #A TCC ACC CAA TTA CTA      48    Gly Trp Gly Ala Val Tyr Glu Gly Gly Ala Gl - #y Ser Thr Gln Leu Leu    #                 15    - TAC TCC CAA TTT GGC GGT GTT GCT CCT AGC AT - #G ATC TGC GCT GGA TTT      96    Tyr Ser Gln Phe Gly Gly Val Ala Pro Ser Me - #t Ile Cys Ala Gly Phe    #             30    #               120AG GAC GCT TGT    Asp Gln Gly Gly Lys Asp Ala Cys    #         40    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 40 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:37:    - Gly Trp Gly Ala Val Tyr Glu Gly Gly Ala Gl - #y Ser Thr Gln Leu Leu    #                 15    - Tyr Ser Gln Phe Gly Gly Val Ala Pro Ser Me - #t Ile Cys Ala Gly Phe    #             30    - Asp Gln Gly Gly Lys Asp Ala Cys    #         40    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 162 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..162    #ID NO:38:(xi) SEQUENCE DESCRIPTION: SEQ    - GGT TGG GGA ACT ACA GAG AGT ACT GAA TCA TC - #A CAC CAC CTG AAA GAA      48    Gly Trp Gly Thr Thr Glu Ser Thr Glu Ser Se - #r His His Leu Lys Glu    #                 15    - GTT GAA GTG AAC GCT GTA TCT AAT AGT GAA TG - #T CAA AGG CCT AAT GAA      96    Val Glu Val Asn Ala Val Ser Asn Ser Glu Cy - #s Gln Arg Pro Asn Glu    #             30    - GAT CTT GCT ACT ATA TCA TCA CAT GAG ATA TG - #T GCA AGC GTT CCT GGT     144    Asp Leu Ala Thr Ile Ser Ser His Glu Ile Cy - #s Ala Ser Val Pro Gly    #         45    # 162              CT TGT    Gly Gly Lys Asp Ser Cys         50    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 54 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:39:    - Gly Trp Gly Thr Thr Glu Ser Thr Glu Ser Se - #r His His Leu Lys Glu    #                 15    - Val Glu Val Asn Ala Val Ser Asn Ser Glu Cy - #s Gln Arg Pro Asn Glu    #             30    - Asp Leu Ala Thr Ile Ser Ser His Glu Ile Cy - #s Ala Ser Val Pro Gly    #         45    - Gly Gly Lys Asp Ser Cys         50    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 40 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:40:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Glu Asn Ala Lys Glu Lys Se - #r Asp Val Pro Tyr Gln    #                15    - Val Ser Leu Arg Asn Ala Glu Asn Lys His Ph - #e Cys Gly Gly Ala Ile    #            30    - Ile Asp Asp Tyr Trp Val Leu Thr    #        40    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 25 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:41:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Se - #r Ala Pro Phe Met Val    #                15    - Ser Leu Gln Ala Glu Asp Tyr Phe His    #            25    - (2) INFORMATION FOR SEQ ID NO:42:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:42:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Ile Gly Gly Glu Val Ala Gly Glu Gly Se - #r Ala Pro Tyr Gln Val    #                15    - Ser Leu Arg Thr Lys Glu Gly Asn His Phe    #            25    - (2) INFORMATION FOR SEQ ID NO:43:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 29 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:43:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Thr Ala Val Asp Ile Arg Gl - #y Phe Pro Gly Arg Tyr    #                15    - Gln Phe Lys Pro Lys Pro Ser Phe Leu Trp Tr - #p Phe Tyr    #            25    - (2) INFORMATION FOR SEQ ID NO:44:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 35 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:44:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Asn Gly Leu Glu Ala Gly Val Gly Gl - #n Phe Pro Ile Gln Val    #                15    - Phe Leu Asp Leu Thr Asn Ile Arg Asp Glu Ly - #s Ser Arg Cys Gly Gly    #            30    - Ala Leu Phe            35    - (2) INFORMATION FOR SEQ ID NO:45:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 24 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:45:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Il - #e Thr Pro Phe Ile Gly    #                15    - Phe Phe Ala Ser Gly Arg Leu Phe                20    - (2) INFORMATION FOR SEQ ID NO:46:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:46:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Val Gly Gly Asn Asp Val Ser Xaa Lys Il - #e Phe Trp Gln Val Ser    #                15    - Ile Gln Ser Asn Xaa Gln His Phe Cys Gly    #            25    - (2) INFORMATION FOR SEQ ID NO:47:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:47:(xi) SEQUENCE DESCRIPTION: SEQ    - Ile Ile Gly Gly Glu Asp Ala Pro Glu Gly Se - #r Ala Pro Tyr Gln Val    #                15    - Ser Leu Arg Asn Gln Asn Leu Glu His Phe Cy - #s Gly Gly Ser Ile    #            30    - (2) INFORMATION FOR SEQ ID NO:48:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (primer)    #ID NO:48:(xi) SEQUENCE DESCRIPTION: SEQ    #          32      CWTT YGATTCWGGW GG    - (2) INFORMATION FOR SEQ ID NO:49:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (primer)    #ID NO:49:(xi) SEQUENCE DESCRIPTION: SEQ    # 20               TATT    - (2) INFORMATION FOR SEQ ID NO:50:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 453 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..453    #ID NO:50:(xi) SEQUENCE DESCRIPTION: SEQ    - CAC GAG TTT TGT GCG AGT GTC AGA TAT TGC AG - #C TCT ATG AGT AAC AAG      48    His Glu Phe Cys Ala Ser Val Arg Tyr Cys Se - #r Ser Met Ser Asn Lys    #                 15    - AAA GGA TTA GTA CTG GGC ATC TAC GAC AAT GA - #A TTC GAT AAA AAA ATA      96    Lys Gly Leu Val Leu Gly Ile Tyr Asp Asn Gl - #u Phe Asp Lys Lys Ile    #             30    - AGG TTA ACG CCA ACT GCT GAA CAA TTC AAT CG - #G CGA TTG CAG GGG CGT     144    Arg Leu Thr Pro Thr Ala Glu Gln Phe Asn Ar - #g Arg Leu Gln Gly Arg    #         45    - TTA CTA GAT CTA ATT CAT TTG AGT GGA CCC AT - #T AAA TTG GGC AAG AGC     192    Leu Leu Asp Leu Ile His Leu Ser Gly Pro Il - #e Lys Leu Gly Lys Ser    #     60    - CGT ATT TTC TGG GAT CTC GAT GAA TTC GGC GC - #A GTT GCA GTT GCA GGT     240    Arg Ile Phe Trp Asp Leu Asp Glu Phe Gly Al - #a Val Ala Val Ala Gly    # 80    - TTG GGA AAT CAC TCC CCC TGC GAA CTC CTG GA - #A GAA CTC GAT GTT TTG     288    Leu Gly Asn His Ser Pro Cys Glu Leu Leu Gl - #u Glu Leu Asp Val Leu    #                 95    - CGC GAA AAT GCC AGA ATA GCT GCC GGT GCT GG - #T TGC CAA GCT CTT GCC     336    Arg Glu Asn Ala Arg Ile Ala Ala Gly Ala Gl - #y Cys Gln Ala Leu Ala    #           110    - GCC GAT GGA ATC ACT ACC ATT AGC GTT GAA GT - #A TGG AGC ACC CGG AGG     384    Ala Asp Gly Ile Thr Thr Ile Ser Val Glu Va - #l Trp Ser Thr Arg Arg    #       125    - CGG CCA TGC GAA GGT GCA ATA CTA TCG ACG TT - #C AAA TTC AGG TCA ACA     432    Arg Pro Cys Glu Gly Ala Ile Leu Ser Thr Ph - #e Lys Phe Arg Ser Thr    #   140    #                 453 AGC GGT    Glu Val Val Gln Cys Ser Gly    145                 1 - #50    - (2) INFORMATION FOR SEQ ID NO:51:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 151 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:51:    - His Glu Phe Cys Ala Ser Val Arg Tyr Cys Se - #r Ser Met Ser Asn Lys    #                 15    - Lys Gly Leu Val Leu Gly Ile Tyr Asp Asn Gl - #u Phe Asp Lys Lys Ile    #             30    - Arg Leu Thr Pro Thr Ala Glu Gln Phe Asn Ar - #g Arg Leu Gln Gly Arg    #         45    - Leu Leu Asp Leu Ile His Leu Ser Gly Pro Il - #e Lys Leu Gly Lys Ser    #     60    - Arg Ile Phe Trp Asp Leu Asp Glu Phe Gly Al - #a Val Ala Val Ala Gly    # 80    - Leu Gly Asn His Ser Pro Cys Glu Leu Leu Gl - #u Glu Leu Asp Val Leu    #                 95    - Arg Glu Asn Ala Arg Ile Ala Ala Gly Ala Gl - #y Cys Gln Ala Leu Ala    #           110    - Ala Asp Gly Ile Thr Thr Ile Ser Val Glu Va - #l Trp Ser Thr Arg Arg    #       125    - Arg Pro Cys Glu Gly Ala Ile Leu Ser Thr Ph - #e Lys Phe Arg Ser Thr    #   140    - Glu Val Val Gln Cys Ser Gly    145                 1 - #50    - (2) INFORMATION FOR SEQ ID NO:52:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 258 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..258    #ID NO:52:(xi) SEQUENCE DESCRIPTION: SEQ    - TCA GCA CTC GTT GCC TTG TCT GCA GCT ATT CC - #T CAC TCC AAC AGA GTC      48    Ser Ala Leu Val Ala Leu Ser Ala Ala Ile Pr - #o His Ser Asn Arg Val    #                 15    - GTT GGA GGA CTG GAA GCT GCA GAG GGT TCT GC - #A CCT TAT CAA GTA TCC      96    Val Gly Gly Leu Glu Ala Ala Glu Gly Ser Al - #a Pro Tyr Gln Val Ser    #             30    - TTG CAA GTT GGC AAC TTC CAC TTC TGT GGT GG - #T TCA ATT CTG AAC GAA     144    Leu Gln Val Gly Asn Phe His Phe Cys Gly Gl - #y Ser Ile Leu Asn Glu    #         45    - TAT TGG GTT TTG ACT GCT GCT CAC TGT TTG GG - #T TAT GAC TTC GAC GTG     192    Tyr Trp Val Leu Thr Ala Ala His Cys Leu Gl - #y Tyr Asp Phe Asp Val    #     60    - GTA GTT GGA ACA AAC AAA CTT GAT CAA CCA GG - #T GAA AGA TAC CTC GTA     240    Val Val Gly Thr Asn Lys Leu Asp Gln Pro Gl - #y Glu Arg Tyr Leu Val    # 80    # 258              TT CAC    Glu Gln Thr Phe Val His                     85    - (2) INFORMATION FOR SEQ ID NO:53:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 86 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:53:    - Ser Ala Leu Val Ala Leu Ser Ala Ala Ile Pr - #o His Ser Asn Arg Val    #                 15    - Val Gly Gly Leu Glu Ala Ala Glu Gly Ser Al - #a Pro Tyr Gln Val Ser    #             30    - Leu Gln Val Gly Asn Phe His Phe Cys Gly Gl - #y Ser Ile Leu Asn Glu    #         45    - Tyr Trp Val Leu Thr Ala Ala His Cys Leu Gl - #y Tyr Asp Phe Asp Val    #     60    - Val Val Gly Thr Asn Lys Leu Asp Gln Pro Gl - #y Glu Arg Tyr Leu Val    # 80    - Glu Gln Thr Phe Val His                     85    - (2) INFORMATION FOR SEQ ID NO:54:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 240 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..240    #ID NO:54:(xi) SEQUENCE DESCRIPTION: SEQ    - TTA GAT GGG CGC ATT GTT GGA GGA CAA GAT GC - #T GAT ATT GCC AAA TAT      48    Leu Asp Gly Arg Ile Val Gly Gly Gln Asp Al - #a Asp Ile Ala Lys Tyr    #                 15    - GGC TAT CAA GCT TCA CTC CAA GTA TTT AAC GA - #A CAT TTC TGT GGA GCT      96    Gly Tyr Gln Ala Ser Leu Gln Val Phe Asn Gl - #u His Phe Cys Gly Ala    #             30    - TCA ATA TTG AAT AAT TAT TGG ATT GTC ACA GC - #A GCT CAT TGC ATA TAT     144    Ser Ile Leu Asn Asn Tyr Trp Ile Val Thr Al - #a Ala His Cys Ile Tyr    #         45    - GAT GAA TTC ACG TAT TCA GTT CGA GTC GGC AC - #C AGT TTC CAA GGA AGA     192    Asp Glu Phe Thr Tyr Ser Val Arg Val Gly Th - #r Ser Phe Gln Gly Arg    #     60    - CGT GGT TCC GTT CAT CCT GTG GCA CAA ATT AT - #C AAG CAT CCT GCA TAC     240    Arg Gly Ser Val His Pro Val Ala Gln Ile Il - #e Lys His Pro Ala Tyr    # 80    - (2) INFORMATION FOR SEQ ID NO:55:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 80 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:55:    - Leu Asp Gly Arg Ile Val Gly Gly Gln Asp Al - #a Asp Ile Ala Lys Tyr    #                 15    - Gly Tyr Gln Ala Ser Leu Gln Val Phe Asn Gl - #u His Phe Cys Gly Ala    #             30    - Ser Ile Leu Asn Asn Tyr Trp Ile Val Thr Al - #a Ala His Cys Ile Tyr    #         45    - Asp Glu Phe Thr Tyr Ser Val Arg Val Gly Th - #r Ser Phe Gln Gly Arg    #     60    - Arg Gly Ser Val His Pro Val Ala Gln Ile Il - #e Lys His Pro Ala Tyr    # 80    - (2) INFORMATION FOR SEQ ID NO:56:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 218 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..216    #ID NO:56:(xi) SEQUENCE DESCRIPTION: SEQ    - AGG GAA CAA AAG CTG GAG CTC CAC CGC GGT GC - #G CCG GCT CTA GAA CTA      48    Arg Glu Gln Lys Leu Glu Leu His Arg Gly Al - #a Pro Ala Leu Glu Leu    #                 15    - GTG GAT CCC CCG GGT CTG CAG GAA TTG GCA CG - #A GGA TGT TCT TGG CTG      96    Val Asp Pro Pro Gly Leu Gln Glu Leu Ala Ar - #g Gly Cys Ser Trp Leu    #             30    - TGT TTA GTA GCT ATT CTT TGT GCA GTG GCT GC - #T GGG CCT ACT AAT CGC     144    Cys Leu Val Ala Ile Leu Cys Ala Val Ala Al - #a Gly Pro Thr Asn Arg    #         45    - ATT GTT GGA GGA TTG GAG GCG AAA AAT GGA AT - #C ACC CCA TTC ATC GGT     192    Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Il - #e Thr Pro Phe Ile Gly    #     60    #             218  GA AGA CTA TTT CA    Phe Phe Ala Ser Gly Arg Leu Phe    # 70    - (2) INFORMATION FOR SEQ ID NO:57:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 72 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:57:    - Arg Glu Gln Lys Leu Glu Leu His Arg Gly Al - #a Pro Ala Leu Glu Leu    #                 15    - Val Asp Pro Pro Gly Leu Gln Glu Leu Ala Ar - #g Gly Cys Ser Trp Leu    #             30    - Cys Leu Val Ala Ile Leu Cys Ala Val Ala Al - #a Gly Pro Thr Asn Arg    #         45    - Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Il - #e Thr Pro Phe Ile Gly    #     60    - Phe Phe Ala Ser Gly Arg Leu Phe    # 70    - (2) INFORMATION FOR SEQ ID NO:58:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 240 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..240    #ID NO:58:(xi) SEQUENCE DESCRIPTION: SEQ    - ACG AGG TTT CGC TTA GCA ATT GTA TGT GCT CT - #C GCT GTC TGC ACA TTC      48    Thr Arg Phe Arg Leu Ala Ile Val Cys Ala Le - #u Ala Val Cys Thr Phe    #                 15    - GGT GCC AGT GTT CCA GAA CCA TGG AAA AGA TT - #A GAT GGT AGA ATC GTA      96    Gly Ala Ser Val Pro Glu Pro Trp Lys Arg Le - #u Asp Gly Arg Ile Val    #             30    - GGA GGA CAC GAT ACC AGC ATC GAT AAA CAC CC - #T CAT CAA GTA TCT TTA     144    Gly Gly His Asp Thr Ser Ile Asp Lys His Pr - #o His Gln Val Ser Leu    #         45    - TTG TAC TCC AGC CAC AAT TGT GGT GGT TCC TT - #G ATT GCC AAA AAC TGG     192    Leu Tyr Ser Ser His Asn Cys Gly Gly Ser Le - #u Ile Ala Lys Asn Trp    #     60    - GTT TTG ACT GCA GCT CAT TGC ATT GGA GTT AA - #C AAA TAC AAT GTC CGT     240    Val Leu Thr Ala Ala His Cys Ile Gly Val As - #n Lys Tyr Asn Val Arg    # 80    - (2) INFORMATION FOR SEQ ID NO:59:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 80 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:59:    - Thr Arg Phe Arg Leu Ala Ile Val Cys Ala Le - #u Ala Val Cys Thr Phe    #                 15    - Gly Ala Ser Val Pro Glu Pro Trp Lys Arg Le - #u Asp Gly Arg Ile Val    #             30    - Gly Gly His Asp Thr Ser Ile Asp Lys His Pr - #o His Gln Val Ser Leu    #         45    - Leu Tyr Ser Ser His Asn Cys Gly Gly Ser Le - #u Ile Ala Lys Asn Trp    #     60    - Val Leu Thr Ala Ala His Cys Ile Gly Val As - #n Lys Tyr Asn Val Arg    # 80    - (2) INFORMATION FOR SEQ ID NO:60:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 234 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..234    #ID NO:60:(xi) SEQUENCE DESCRIPTION: SEQ    - CCC TCA CTA AAG GGA ACA AAA GCT GGA GCT CC - #A CCG CGG TGC GCC GCT      48    Pro Ser Leu Lys Gly Thr Lys Ala Gly Ala Pr - #o Pro Arg Cys Ala Ala    #                 15    - CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG AA - #T TCG GCA CGA GCG TTT      96    Leu Glu Leu Val Asp Pro Pro Gly Cys Arg As - #n Ser Ala Arg Ala Phe    #             30    - GGT TGG ATT GAG CGC GTC TCA TCT TAC AAG AT - #A AAG GAT AGA TTA GAT     144    Gly Trp Ile Glu Arg Val Ser Ser Tyr Lys Il - #e Lys Asp Arg Leu Asp    #         45    - GGG CGC ATT GTT GGA GGA CAA GAT GCT GAT AT - #T GCC AAA TAT GGC TAT     192    Gly Arg Ile Val Gly Gly Gln Asp Ala Asp Il - #e Ala Lys Tyr Gly Tyr    #     60    - CAA GCT TCA CTC CAA GTA CTT AAC GAA CAT TT - #C TGT GGA GCT    # 234    Gln Ala Ser Leu Gln Val Leu Asn Glu His Ph - #e Cys Gly Ala    # 75    - (2) INFORMATION FOR SEQ ID NO:61:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 78 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:61:    - Pro Ser Leu Lys Gly Thr Lys Ala Gly Ala Pr - #o Pro Arg Cys Ala Ala    #                 15    - Leu Glu Leu Val Asp Pro Pro Gly Cys Arg As - #n Ser Ala Arg Ala Phe    #             30    - Gly Trp Ile Glu Arg Val Ser Ser Tyr Lys Il - #e Lys Asp Arg Leu Asp    #         45    - Gly Arg Ile Val Gly Gly Gln Asp Ala Asp Il - #e Ala Lys Tyr Gly Tyr    #     60    - Gln Ala Ser Leu Gln Val Leu Asn Glu His Ph - #e Cys Gly Ala    # 75    - (2) INFORMATION FOR SEQ ID NO:62:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 291 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..291    #ID NO:62:(xi) SEQUENCE DESCRIPTION: SEQ    - GCG GTG ATT GTG TCA TTT GTT CTG GCT TGT GC - #A TTT TCT GTA CAG GCT      48    Ala Val Ile Val Ser Phe Val Leu Ala Cys Al - #a Phe Ser Val Gln Ala    #                 15    - CTT CCA TCA AGC AGA ATT GTC AAT GGA CTT GA - #A GCA GGA GTT GGA CAA      96    Leu Pro Ser Ser Arg Ile Val Asn Gly Leu Gl - #u Ala Gly Val Gly Gln    #             30    - TTT CCA ATT CAG GTT TTC TTA GAC TTG ACA AA - #T ATC AGA GAC GAA AAA     144    Phe Pro Ile Gln Val Phe Leu Asp Leu Thr As - #n Ile Arg Asp Glu Lys    #         45    - TCC AGA TGT GGT GGT GCT TTG TTA TCA GAT TC - #A TGG GTT TTG ACT GCT     192    Ser Arg Cys Gly Gly Ala Leu Leu Ser Asp Se - #r Trp Val Leu Thr Ala    #     60    - GCT CAT TGT TTT GAT GAT TTG AAG TCT ATG GT - #A GTG TCC GTT GGT GCT     240    Ala His Cys Phe Asp Asp Leu Lys Ser Met Va - #l Val Ser Val Gly Ala    # 80    - CAT GAT GTC AGC AAA TCT GAA GAA CCT CAC AG - #G CAA ACC AGG AAA CCT     288    His Asp Val Ser Lys Ser Glu Glu Pro His Ar - #g Gln Thr Arg Lys Pro    #                 95    #            291    Glu    - (2) INFORMATION FOR SEQ ID NO:63:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 97 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:63:    - Ala Val Ile Val Ser Phe Val Leu Ala Cys Al - #a Phe Ser Val Gln Ala    #                 15    - Leu Pro Ser Ser Arg Ile Val Asn Gly Leu Gl - #u Ala Gly Val Gly Gln    #             30    - Phe Pro Ile Gln Val Phe Leu Asp Leu Thr As - #n Ile Arg Asp Glu Lys    #         45    - Ser Arg Cys Gly Gly Ala Leu Leu Ser Asp Se - #r Trp Val Leu Thr Ala    #     60    - Ala His Cys Phe Asp Asp Leu Lys Ser Met Va - #l Val Ser Val Gly Ala    # 80    - His Asp Val Ser Lys Ser Glu Glu Pro His Ar - #g Gln Thr Arg Lys Pro    #                 95    - Glu    - (2) INFORMATION FOR SEQ ID NO:64:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 144 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..144    #ID NO:64:(xi) SEQUENCE DESCRIPTION: SEQ    - GTA CTG ATC GTT TTA GCA GTC ATT GAA TTC GC - #A TCA GCG TCT TCA ATC      48    Val Leu Ile Val Leu Ala Val Ile Glu Phe Al - #a Ser Ala Ser Ser Ile    #                 15    - GGC TGG AGA ATC GTG GGT GGT GAA AAT GCT AA - #A GAA AAA TCG GTG CCC      96    Gly Trp Arg Ile Val Gly Gly Glu Asn Ala Ly - #s Glu Lys Ser Val Pro    #             30    - TAT CAA GTT TCM CTT CGA AAT GCT GAA AAC AA - #A CAT TTY TGT GGR GGR     144    Tyr Gln Val Ser Leu Arg Asn Ala Glu Asn Ly - #s His Phe Cys Gly Gly    #         45    - (2) INFORMATION FOR SEQ ID NO:65:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 48 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:65:    - Val Leu Ile Val Leu Ala Val Ile Glu Phe Al - #a Ser Ala Ser Ser Ile    #                 15    - Gly Trp Arg Ile Val Gly Gly Glu Asn Ala Ly - #s Glu Lys Ser Val Pro    #             30    - Tyr Gln Val Ser Leu Arg Asn Ala Glu Asn Ly - #s His Phe Cys Gly Gly    #         45    - (2) INFORMATION FOR SEQ ID NO:66:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 390 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..390    #ID NO:66:(xi) SEQUENCE DESCRIPTION: SEQ    - TTC GGC TTC AAG CTA AGT CAT TTG GTA AGT AA - #G TAC TGT GCT TGT GCA      48    Phe Gly Phe Lys Leu Ser His Leu Val Ser Ly - #s Tyr Cys Ala Cys Ala    #                 15    - TTA GCA TCG GCA CTG AAG TAC TCC ATC GAT CA - #T GGT CCT CGT ATC ATC      96    Leu Ala Ser Ala Leu Lys Tyr Ser Ile Asp Hi - #s Gly Pro Arg Ile Ile    #             30    - GGA GGT GAA GTT GCA GGT GAA GGA TCA GCA CC - #T TAC CAG GTG TCC TTA     144    Gly Gly Glu Val Ala Gly Glu Gly Ser Ala Pr - #o Tyr Gln Val Ser Leu    #         45    - AGA ACC AAG GAA GGA AAT CAT TTT TGC GGT GG - #A TCA ATA CTA AAT AAG     192    Arg Thr Lys Glu Gly Asn His Phe Cys Gly Gl - #y Ser Ile Leu Asn Lys    #     60    - CGA TGG GTT GTA ACT GCA GCA CAT TGT CTT GA - #A CCG GAA ATA TTA GAT     240    Arg Trp Val Val Thr Ala Ala His Cys Leu Gl - #u Pro Glu Ile Leu Asp    # 80    - TCG GTA TAC GTC GGA TCC AAT CAC TTA GAC CG - #A AAA GGC AGA TAT TAC     288    Ser Val Tyr Val Gly Ser Asn His Leu Asp Ar - #g Lys Gly Arg Tyr Tyr    #                 95    - GAC GTA GAA CGG TAT ATA ATT CAT GAA AAA TA - #T ATA GGA GAA CTA AAT     336    Asp Val Glu Arg Tyr Ile Ile His Glu Lys Ty - #r Ile Gly Glu Leu Asn    #           110    - AAT TTT TAT GCT GAC ATC GGT CTA ATA AAA CT - #T GAT GGA AGA CTT AGA     384    Asn Phe Tyr Ala Asp Ile Gly Leu Ile Lys Le - #u Asp Gly Arg Leu Arg    #       125    #          390    Ile Gln        130    - (2) INFORMATION FOR SEQ ID NO:67:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 130 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:67:    - Phe Gly Phe Lys Leu Ser His Leu Val Ser Ly - #s Tyr Cys Ala Cys Ala    #                 15    - Leu Ala Ser Ala Leu Lys Tyr Ser Ile Asp Hi - #s Gly Pro Arg Ile Ile    #             30    - Gly Gly Glu Val Ala Gly Glu Gly Ser Ala Pr - #o Tyr Gln Val Ser Leu    #         45    - Arg Thr Lys Glu Gly Asn His Phe Cys Gly Gl - #y Ser Ile Leu Asn Lys    #     60    - Arg Trp Val Val Thr Ala Ala His Cys Leu Gl - #u Pro Glu Ile Leu Asp    # 80    - Ser Val Tyr Val Gly Ser Asn His Leu Asp Ar - #g Lys Gly Arg Tyr Tyr    #                 95    - Asp Val Glu Arg Tyr Ile Ile His Glu Lys Ty - #r Ile Gly Glu Leu Asn    #           110    - Asn Phe Tyr Ala Asp Ile Gly Leu Ile Lys Le - #u Asp Gly Arg Leu Arg    #       125    - Ile Gln        130    - (2) INFORMATION FOR SEQ ID NO:68:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 240 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..240    -     (ix) FEATURE:    #any amino acidAME/KEY: Xaa =              (B) LOCATION: 73    #ID NO:68:(xi) SEQUENCE DESCRIPTION: SEQ    - CGG GCT GCA GGA ATT CGG CAC GAG AAG AAA CT - #G CCA ATA TTA ATC GCC      48    Arg Ala Ala Gly Ile Arg His Glu Lys Lys Le - #u Pro Ile Leu Ile Ala    #                 15    - TTG ATC GGA TGC GTT CTT TCT GAA GAA ATA GA - #G GAT CGC ATT GTC GGC      96    Leu Ile Gly Cys Val Leu Ser Glu Glu Ile Gl - #u Asp Arg Ile Val Gly    #             30    - GGA ACG GCA GTT GAT ATA AGA GGT TTT CCC TG - #G CAG GTA TCA ATT CAA     144    Gly Thr Ala Val Asp Ile Arg Gly Phe Pro Tr - #p Gln Val Ser Ile Gln    #         45    - ACC GAA AAC CGT CAT TTT TGT GGT GGT TCT AT - #T ATC GAT AAA AGC TGG     192    Thr Glu Asn Arg His Phe Cys Gly Gly Ser Il - #e Ile Asp Lys Ser Trp    #     60    - ATA TTA ACT GCC GCA CAT TGT GTA CMC GAT AT - #G AAG ATG TCG AAC TGG     240    Ile Leu Thr Ala Ala His Cys Val Xaa Asp Me - #t Lys Met Ser Asn Trp    # 80    - (2) INFORMATION FOR SEQ ID NO:69:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 80 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:    #any amino acidAME/KEY: Xaa =              (B) LOCATION: 73    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:69:    - Arg Ala Ala Gly Ile Arg His Glu Lys Lys Le - #u Pro Ile Leu Ile Ala    #                 15    - Leu Ile Gly Cys Val Leu Ser Glu Glu Ile Gl - #u Asp Arg Ile Val Gly    #             30    - Gly Thr Ala Val Asp Ile Arg Gly Phe Pro Tr - #p Gln Val Ser Ile Gln    #         45    - Thr Glu Asn Arg His Phe Cys Gly Gly Ser Il - #e Ile Asp Lys Ser Trp    #     60    - Ile Leu Thr Ala Ala His Cys Val His Asp Me - #t Lys Met Ser Asn Trp    # 80    - (2) INFORMATION FOR SEQ ID NO:70:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 177 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..177    #ID NO:70:(xi) SEQUENCE DESCRIPTION: SEQ    - CAC GAG ATT TTA TTA AGC GCA TTA TTT GCA AG - #T GTA ATT TGC TCC TTT      48    His Glu Ile Leu Leu Ser Ala Leu Phe Ala Se - #r Val Ile Cys Ser Phe    #                 15    - AAC GCG GAA GTA CAA AAT CGA ATC GTT GGT GG - #C AAT GAT GTA AGT ATT      96    Asn Ala Glu Val Gln Asn Arg Ile Val Gly Gl - #y Asn Asp Val Ser Ile    #             30    - TCA AAA ATT GGG TGG CAA GTA TCT ATT CAA AG - #T AAT AAA CAA CAT TTC     144    Ser Lys Ile Gly Trp Gln Val Ser Ile Gln Se - #r Asn Lys Gln His Phe    #         45    #        177T TCA ATC ATT GCT AAA GAT GGG TC - #C    Cys Gly Gly Ser Ile Ile Ala Lys Asp Gly Se - #r    #     55    - (2) INFORMATION FOR SEQ ID NO:71:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 59 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:71:    - His Glu Ile Leu Leu Ser Ala Leu Phe Ala Se - #r Val Ile Cys Ser Phe    #                 15    - Asn Ala Glu Val Gln Asn Arg Ile Val Gly Gl - #y Asn Asp Val Ser Ile    #             30    - Ser Lys Ile Gly Trp Gln Val Ser Ile Gln Se - #r Asn Lys Gln His Phe    #         45    - Cys Gly Gly Ser Ile Ile Ala Lys Asp Gly Se - #r    #     55    - (2) INFORMATION FOR SEQ ID NO:72:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 168 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..168    #ID NO:72:(xi) SEQUENCE DESCRIPTION: SEQ    - ATC ATG GCA AAT TTT AGG CTA TTC ACC TTA CT - #A GCC TTG GTT TCA GTA      48    Ile Met Ala Asn Phe Arg Leu Phe Thr Leu Le - #u Ala Leu Val Ser Val    #                 15    - GCA ACT TCC AAA TAT ATT GAT CCA AGA ATA AT - #T GGA GGC GAA GAT GCT      96    Ala Thr Ser Lys Tyr Ile Asp Pro Arg Ile Il - #e Gly Gly Glu Asp Ala    #             30    - CCT GAA GGC TCG GCT CCG TAC CAA GTT TCA TT - #G AGA AAT CAG AAT CTG     144    Pro Glu Gly Ser Ala Pro Tyr Gln Val Ser Le - #u Arg Asn Gln Asn Leu    #         45    #               168GT GGT TCC ATT    Glu His Phe Cys Gly Gly Ser Ile    #     55    - (2) INFORMATION FOR SEQ ID NO:73:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 56 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:73:    - Ile Met Ala Asn Phe Arg Leu Phe Thr Leu Le - #u Ala Leu Val Ser Val    #                 15    - Ala Thr Ser Lys Tyr Ile Asp Pro Arg Ile Il - #e Gly Gly Glu Asp Ala    #             30    - Pro Glu Gly Ser Ala Pro Tyr Gln Val Ser Le - #u Arg Asn Gln Asn Leu    #         45    - Glu His Phe Cys Gly Gly Ser Ile    #     55    - (2) INFORMATION FOR SEQ ID NO:74:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 192 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..192    #ID NO:74:(xi) SEQUENCE DESCRIPTION: SEQ    - GCA CGA GAT CGC ATT GTT GGA GGA TTG GAG GC - #G AAA AAT GGA TCA GCC      48    Ala Arg Asp Arg Ile Val Gly Gly Leu Glu Al - #a Lys Asn Gly Ser Ala    #                 15    - CCA TTC ATG GTT TCT TTG CAA GCG GAA GAC TA - #T TTT CAT TTT TGT GGA      96    Pro Phe Met Val Ser Leu Gln Ala Glu Asp Ty - #r Phe His Phe Cys Gly    #             30    - TCC TCT ATT CTG AAT GAG AGA TGG GTT CTT AC - #T GCT GCT CAC TGT ATC     144    Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Th - #r Ala Ala His Cys Ile    #         45    - CAA CCA AAT GTA CAC AAG TAC GTT TAC GTC GG - #T TCG AAC AAC GTA GAA     192    Gln Pro Asn Val His Lys Tyr Val Tyr Val Gl - #y Ser Asn Asn Val Glu    #     60    - (2) INFORMATION FOR SEQ ID NO:75:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 64 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:75:    - Ala Arg Asp Arg Ile Val Gly Gly Leu Glu Al - #a Lys Asn Gly Ser Ala    #                 15    - Pro Phe Met Val Ser Leu Gln Ala Glu Asp Ty - #r Phe His Phe Cys Gly    #             30    - Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Th - #r Ala Ala His Cys Ile    #         45    - Gln Pro Asn Val His Lys Tyr Val Tyr Val Gl - #y Ser Asn Asn Val Glu    #     60    - (2) INFORMATION FOR SEQ ID NO:76:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 207 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..204    #ID NO:76:(xi) SEQUENCE DESCRIPTION: SEQ    - CCA ATC CAC GAT AGC CAA TAT GCA CTT TTG CA - #G ATA TGG GTC AAG GGT      48    Pro Ile His Asp Ser Gln Tyr Ala Leu Leu Gl - #n Ile Trp Val Lys Gly    #                 15    - GCA TGT AAG GGT GAT TCC GGT GGC CCC TTA GT - #C ATC AAT GGA CAA CTT      96    Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Va - #l Ile Asn Gly Gln Leu    #             30    - CAT GGA ATT GTT TCC TGG GGC ATT CCT TGC GC - #T GTC GCA AGC CTG ATG     144    His Gly Ile Val Ser Trp Gly Ile Pro Cys Al - #a Val Ala Ser Leu Met    #         45    - TAT TCA CAA GAG TTT CTC ATT ATG TCG ATT GG - #A TTA AAT CCA AAA TTG     192    Tyr Ser Gln Glu Phe Leu Ile Met Ser Ile Gl - #y Leu Asn Pro Lys Leu    #     60    #   207            AG    Asn Lys Ile Val     65    - (2) INFORMATION FOR SEQ ID NO:77:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 68 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:77:    - Pro Ile His Asp Ser Gln Tyr Ala Leu Leu Gl - #n Ile Trp Val Lys Gly    #                 15    - Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Va - #l Ile Asn Gly Gln Leu    #             30    - His Gly Ile Val Ser Trp Gly Ile Pro Cys Al - #a Val Ala Ser Leu Met    #         45    - Tyr Ser Gln Glu Phe Leu Ile Met Ser Ile Gl - #y Leu Asn Pro Lys Leu    #     60    - Asn Lys Ile Val     65    - (2) INFORMATION FOR SEQ ID NO:78:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 159 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..156    #ID NO:78:(xi) SEQUENCE DESCRIPTION: SEQ    - GGA GGT CCT TTG GCA ATC AAT GGT GAA CTT GT - #T GGT GTT ACT TCA TTC      48    Gly Gly Pro Leu Ala Ile Asn Gly Glu Leu Va - #l Gly Val Thr Ser Phe    #                 15    - ATT ATG GGG ACA TGT GGA GGA GGA CAT CCT GA - #T GTC TTC GGT CGA GTC      96    Ile Met Gly Thr Cys Gly Gly Gly His Pro As - #p Val Phe Gly Arg Val    #             30    - CTT GAC TTC AAA CCA TGG ATT GAT TCT CAT AT - #G GCA AAT GAC GGC GCT     144    Leu Asp Phe Lys Pro Trp Ile Asp Ser His Me - #t Ala Asn Asp Gly Ala    #         45    #   159            AA    Asn Ser Phe Ile         50    - (2) INFORMATION FOR SEQ ID NO:79:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 52 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:79:    - Gly Gly Pro Leu Ala Ile Asn Gly Glu Leu Va - #l Gly Val Thr Ser Phe    #                 15    - Ile Met Gly Thr Cys Gly Gly Gly His Pro As - #p Val Phe Gly Arg Val    #             30    - Leu Asp Phe Lys Pro Trp Ile Asp Ser His Me - #t Ala Asn Asp Gly Ala    #         45    - Asn Ser Phe Ile         50    __________________________________________________________________________

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

What is claimed is:
 1. An isolated flea aminopeptidase nucleic acidmolecule comprising a flea nucleic acid sequence selected from the groupconsisting of: (a) nucleic acid sequence SEQ ID NO:50; (b) a homologueof said nucleic acid sequence SEQ ID NO:50 containing one or morenucleotide insertions, deletions, or substitutions, wherein saidhomologue encodes at least one epitope that elicits an immune responseagainst a protein comprising amino acid sequence SEQ ID NO;51 and (c)the complement of a nucleic acid molecule of (a) or (b), and whereinsaid nucleic acid molecule of (a), (b) or (c) is at least 18 nucleotidesin length.
 2. The nucleic acid molecule of claim 1, wherein said nucliecacid molecule comprises the nucleic acid molecule nfAP₄₅₃ or naturallyocurring allelic variants thereof.
 3. The nucleic acid molecule of claim1, wherein said nucliec acid molecule comprises a nucleic acid sequencethat encodes a protein having the amino acid sequence SEQ ID NO;51 ornaturally ocurring allelic variants thereof.
 4. The nucleic acidmolecule of claim 1, wherein an aminopeptidase protein encoded by saidisolated flea aminopeptidase nucleic acid molecule has aminopeptidaseactivity.
 5. A recombinant molecule comprising a nucleic acid moleculeas set forth in claim 1 operatively linked to a transcription controlsequence.
 6. A recombinant virus comprising a recombinant molecule asset forth in claim
 5. 7. An isolated recombinant cell comprising anucleic acid molecule as set forth in claim 1, said cell being capableof expressing said nucleic acid molecule.
 8. A method to produce a fleaaminopeptidase protein, said method comprising culturing a cell capableof expressing said protein, said protein being encoded by a nucleic acidmolecule comprising a nucleic acid sequence selected from the groupconsisting of: (a) nucleic acid sequence SEQ ID NO:50; and (b) ahomologue of said nucleic acid sequence SEQ ID NO:50 containing one ormore nucleotide insertions, deletions, or substitutions, wherein saidhomologue encodes at least one epitope that elicits an immune responseagainst a protein comprising amino acid sequence SEQ ID NO:51 andwherein said nucleic acid molecule of (a) or (b) is at least 18nucleotides in length and is from flea.